PRLR-specific antibody and uses thereof

ABSTRACT

PRLR-specific antibodies are provided, along with pharmaceutical compositions containing such antibody, kits containing a pharmaceutical composition, and methods of preventing and treating cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/946,360 filed Jun. 26, 2007 and U.S. Provisional PatentApplication No. 60/838,648 filed Aug. 18, 2006.

TECHNICAL FIELD

This invention relates to methods for preventing and treating cancer byadministering PRLR-specific antibodies.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of death in the United States.Although “cancer” is used to describe many different types of cancer,i.e. breast, prostate, lung, colon, pancreas, each type of cancerdiffers both at the phenotypic level and the genetic level. Theunregulated growth characteristic of cancer occurs when the expressionof one or more genes becomes dysregulated due to mutations, and cellgrowth can no longer be controlled.

Genes are often classified in two classes, oncogenes and tumorsuppressor genes. Oncogenes are genes whose normal function is topromote cell growth, but only under specific conditions. When anoncogene gains a mutation and then loses that control, it promotesgrowth under all conditions. However, it has been found that for cancerto be truly successful the cancer must also acquire mutations in tumorsuppressor genes. The normal function of tumor suppressor genes is tostop cellular growth. Examples of tumor suppressors include p53, p16,p21, and APC, all of which, when acting normally, stop a cell fromdividing and growing uncontrollably. When a tumor suppressor is mutatedor lost, that brake on cellular growth is also lost, allowing cells tonow grow without restraints.

Prolactin receptor (PRLR) is a single membrane-spanning class 1 cytokinereceptor that is homologous to receptors for members of the cytokinesuperfamily, such as the receptors for IL2, IL3, IL4, IL6, IL7,erythropoietin, and GM-CSF. PRLR is involved in multiple biologicalfunctions, including cell growth, differentiation, development,lactation and reproduction. It has no intrinsic tyrosine kinaseactivity; ligand binding leads to receptor dimerization,cross-phosphorylation of Jak2 and downstream signaling. Human prolactinreceptor cDNA was originally isolated from hepatoma and breast cancerlibraries (Boutin, J.-M. et al., Molec. Endocr. 3: 1455-1461, 1989). Thenucleotide sequence predicted a mature protein of 598 amino acids with amuch longer cytoplasmic domain than the rat liver PRL receptor. Theprolactin receptor gene resides in the same chromosomal region as thegrowth hormone receptor gene, which has been mapped to 5; 13-p12 (Arden,K. C. et al. Cytogenet. Cell Gene 53: 161-165, 1990; Arden, K. C. etal., (Abstract) AM. J. Hum. Genet. 45 (suppl.): A129 only, 1989). Growthhormone also binds to the prolactin receptor and activates the receptor.

The genomic organization of the human PRLR gene has been determined (Hu,Z.-Z. et al., J. Clin. Endocr. Metab. 84: 1153-1156, 1999). The5-prime-untranslated region of the PRLR gene contains 2 alternativefirst exons: E13, the human counterpart of the rat and mouse E13, and anovel human type of alternative first exon termed E1N. The5-prime-untranslated region also contains a common noncoding exon 2 andpart of exon 3, which contains the translation initiation codon. The E13and E1N exons are within 800 basepairs of each other. These 2 exons areexpressed in human breast tissue, breast cancer cells, gonads, andliver. Overall, the transcript containing E13 is prevalent in mosttissues. The PRLR gene product is encoded by exons 3-10, of which exon10 encodes most of the intracellular domain. The E13 and E1N exons aretranscribed from alternative promoters PIII and PN, respectively. ThePIII promoter contains Sp1 and C/EBP elements that are identical tothose in the rodent promoter and is 81% similar to the region −480/−106in the rat and mouse. The PN promoter contains putative binding sitesfor ETS family proteins and a half-site for nuclear receptors.

PRLR exists in a number of different isoforms that differ in the lengthof their cytoplasmic domains. Four PRLR mRNA isoforms (L, I, S1a, andS1b) have been demonstrated in human subcutaneous abdominal adiposetissue and breast adipose tissue (Ling, C. et al., J. Clin. Endocr.Metab. 88: 1804-1808, 2003). In addition, they detected L-PRLR andI-PRLR protein expression in human subcutaneous abdominal adipose tissueand breast adipose tissue using immunoblot analysis. PRL reduced thelipoprotein lipase activity in human adipose tissue compared withcontrol. Ling et al. suggest that these results demonstrated a directeffect of PRL, via functional PRLRs, in reducing the LPL activity inhuman adipose tissue, and that these results suggested that LPL mightalso be regulated in this fashion during lactation. The function ofthese PRLR isoforms in rat has been elucidated (Perrot-Applanat, M. etal., Molec. Endocr. 11: 1020-1032, 1997). Like the known long form (591amino acids), the Nb2 form, which lacks 198 amino acids of thecytoplasmic domain, is able to transmit a lactogenic signal. Incontrast, the short form, which lacks 291 amino acids of the cytoplasmicdomain, is inactive. The function of the short form was examined aftercotransfection of both the long and short forms. These results show thatthe short form acts as a dominant-negative inhibitor through theformation of inactive heterodimers, resulting in the inhibition of Januskinase 2 activation. Perrot-Applanat et al. suggest thatheterodimerization of PRLR can positively or negatively activate PRLtranscription.

Recent reports have suggested that PRLR is over-expressed in humanbreast cancer and prostate cancer tissues (Li et al., Cancer Res.,64:4774-4782, 2004; Gill et al., J Clin Pathol., 54:956-960, 2001;Touraine et al., J Clin Endocrinol Metab., 83:667-674, 1998). Li et al.,reported that Stat5 activation and PRLR expression is associated withhigh histological grade in 54% of prostate cancer specimens (Li et al.,supra). Other reports have suggested that primary breast cancerspecimens are responsive to PRL in colony formation assays and thatplasma PRL concentrations correlate with breast cancer risk (Tworoger etal., Cancer Res., 64:6814-6819, 2004; Tworoger et al., Cancer Res.,66:2476-2482, 2006). Another report indicated that PRL transgenic micedevelop malignant mammary carcinomas or prostate hyperplasia (Wennbo etal., J Clin Invest., 100:2744-2751, 1997; Wennbo et al., Endocrinology,138:4410-4415, 1997).

A PRLR monoclonal antibody diminished the incidence of mammary tumors inmice (Sissom et al., Am. J. Pathol. 133:589-595, 1988). In addition, aPRL antagonist (S179D mutant PRL) inhibited proliferation of a humanprostate carcinoma cell line, DU-145, in vitro and DU-145 induced tumorsin vivo (Xu et al., Cancer Res., 61:6098-6104, 2001).

Thus, there is a need to identify compositions and methods that modulatePRLR and its role in such cancers. The present invention is directed tothese, as well as other, important needs.

SUMMARY OF THE INVENTION

The nucleotide sequence for PRLR is set out in SEQ ID NO: 1, and theamino acid sequence is set out in SEQ ID NO: 2. The extracellular domain(ECD) consists of amino acids 25 through 234 of SEQ ID NO: 2, which canbe divided into two major domains, S1 (amino acids 25-122) and S2 (aminoacids 123-234). A number of different isoforms of PRLR have beenidentified: long (L), intermediate (I), ΔS1, an inactive soluble form(PRLBP), and inactive short forms S1a and S1b. The exons and nucleotideregions contained within each isoform are displayed in FIG. 1. Inexemplary embodiments, the invention contemplates antibodies that bindto the S1 domain and/or to the S2 domain. Such antibodies that bind tothe S2 domain may target all active isoforms. The invention alsocontemplates antibodies that bind specifically to one isoform and notanother (e.g. intermediate and not S1a or S1b), or to the activeisoforms (long, intermediate and ΔS1) but not to the inactive isoforms(S1a and S1b).

The materials and methods of the present invention fulfill theaforementioned and other related needs in the art.

In one embodiment, an antibody that binds the extracellular domain ofPRLR with an equilibrium dissociation constant (K_(D)) of 10⁻⁶ M orlower and competes with any of antibodies chXHA.06.642, chXHA.06.275,he.06.642-1, he.06.642-2, he.06.275-1, he.06.275-2, he.06.275-3,he.06.275-4, XPA.06.128, XPA.06.129, XPA.06.130, XPA.06.131, XPA.06.141,XPA.06.147, XPA.06.148, XPA.06.158, XPA.06.159, XPA.06.163, XPA.06.167,XPA.06.171, XPA.06.178, XPA.06.181, XPA.06.192, XPA.06.202, XPA.06.203,XPA.06.206, XPA.06.207, XPA.06.210, XPA.06.212, XPA.06.217, XPA.06.219,XPA.06.229, XPA.06.233, XPA.06.235, XPA.06.239, XPA.06.145, XHA.06.567,XHA.06.642, XHA.06.983, XHA.06.275, XHA.06.189, or XHA.06.907 forbinding to PRLR by more than 75% is provided. By the term “anequilibrium dissociation constant (K_(D)) of 10⁻⁶ M or lower” it ismeant an equilibrium dissociation constant of, e.g., 10⁻⁶, 10⁻⁷ M, 10⁻⁸M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M or 10⁻¹² M (i.e., a number lower than 10⁻⁶M). In another embodiment, the antibody binds to the same epitope ofPRLR as any of antibodies chXHA.06.642, chXHA.06.275, he.06.642-1,he.06.642-2, he.06.275-1, he.06.275-2, he.06.275-3, he.06.275-4,XPA.06.128, XPA.06.129, XPA.06.130, XPA.06.131, XPA.06.141, XPA.06.147,XPA.06.148, XPA.06.158, XPA.06.159, XPA.06.163, XPA.06.167, XPA.06.171,XPA.06.178, XPA.06.181, XPA.06.192, XPA.06.202, XPA.06.203, XPA.06.206,XPA.06.207, XPA.06.210, XPA.06.212, XPA.06.217, XPA.06.219, XPA.06.229,XPA.06.233, XPA.06.235, XPA.06.239, XPA.06.145, XHA.06.567, XHA.06.642,XHA.06.983, XHA.06.275, XHA.06.189, or XHA.06.907.

In another embodiment, an aforementioned antibody comprises 1, 2, 3, 4,5 or 6 CDRs of any of antibodies chXHA.06.642, chXHA.06.275,he.06.642-1, he.06.642-2, he.06.275-1, he.06.275-2, he.06.275-3,he.06.275-4, XPA.06.128, XPA.06.129, XPA.06.130, XPA.06.131, XPA.06.141,XPA.06.147, XPA.06.148, XPA.06.158, XPA.06.159, XPA.06.163, XPA.06.167,XPA.06.171, XPA.06.178, XPA.06.181, XPA.06.192, XPA.06.202, XPA.06.203,XPA.06.206, XPA.06.207, XPA.06.210, XPA.06.212, XPA.06.217, XPA.06.219,XPA.06.229, XPA.06.233, XPA.06.235, XPA.06.239, XPA.06.145, XHA.06.567,XHA.06.642, XHA.06.983, XHA.06.275, XHA.06.189, or XHA.06.907. Inanother embodiment, an aforementioned antibody is a chimeric antibody, ahumanized antibody, a human engineered antibody, a human antibody, asingle chain antibody or an antibody fragment. In yet anotherembodiment, an aforementioned antibody is provided in which at least oneamino acid within a CDR is substituted by a corresponding residue of acorresponding CDR of another anti-PRLR antibody. In an exemplaryembodiment, an aforementioned antibody is provided in which at least oneamino acid within a CDR from an antibody selected from the groupconsisting of chXHA.06.642, chXHA.06.275, he.06.642-1, he.06.642-2,he.06.275-1, he.06.275-2, he.06.275-3, he.06.275-4, XPA.06.128,XPA.06.129, XPA.06.130, XPA.06.131, XPA.06.141, XPA.06.147, XPA.06.148,XPA.06.158, XPA.06.159, XPA.06.163, XPA.06.167, XPA.06.171, XPA.06.178,XPA.06.181, XPA.06.192, XPA.06.202, XPA.06.203, XPA.06.206, XPA.06.207,XPA.06.210, XPA.06.212, XPA.06.217, XPA.06.219, XPA.06.229, XPA.06.233,XPA.06.235, XPA.06.239, XPA.06.145, XHA.06.567, XHA.06.642, XHA.06.983,XHA.06.275, XHA.06.189, or XHA.06.907 is substituted by a correspondingresidue of a corresponding CDR of another anti-PRLR antibody. In anotherexemplary embodiment, an aforementioned antibody is provided in which atleast one amino acid within a CDR from an antibody selected from thegroup consisting of chXHA.06.642, chXHA.06.275, he.06.642-1,he.06.642-2, he.06.275-1, he.06.275-2, he.06.275-3, he.06.275-4,XPA.06.128, XPA.06.129, XPA.06.130, XPA.06.131, XPA.06.141, XPA.06.147,XPA.06.148, XPA.06.158, XPA.06.159, XPA.06.163, XPA.06.167, XPA.06.171,XPA.06.178, XPA.06.181, XPA.06.192, XPA.06.202, XPA.06.203, XPA.06.206,XPA.06.207, XPA.06.210, XPA.06.212, XPA.06.217, XPA.06.219, XPA.06.229,XPA.06.233, XPA.06.235, XPA.06.239, XPA.06.145, XHA.06.567, XHA.06.642,XHA.06.983, XHA.06.275, XHA.06.189, or XHA.06.907 is substituted by acorresponding residue of a corresponding CDR of another antibodyselected from the group consisting of chXHA.06.642, chXHA.06.275,he.06.642-1, he.06.642-2, he.06.275-1, he.06.275-2, he.06.275-3,he.06.275-4, XPA.06.128, XPA.06.129, XPA.06.130, XPA.06.131, XPA.06.141,XPA.06.147, XPA.06.148, XPA.06.158, XPA.06.159, XPA.06.163, XPA.06.167,XPA.06.171, XPA.06.178, XPA.06.181, XPA.06.192, XPA.06.202, XPA.06.203,XPA.06.206, XPA.06.207, XPA.06.210, XPA.06.212, XPA.06.217, XPA.06.219,XPA.06.229, XPA.06.233, XPA.06.235, XPA.06.239, XPA.06.145, XHA.06.567,XHA.06.642, XHA.06.983, XHA.06.275, XHA.06.189, or XHA.06.907. In stillanother embodiment, an aforementioned antibody is provided in which oneor two amino acids within a CDR have been modified.

In another embodiment of the invention, an aforementioned antibody isprovided that retains at least 60, 65, 70, 75, 80, 85, 90, 91, 92, 93,94, 95, 96, 97, 98, or 99% identity over either the variable light orheavy region to the antibodies of chXHA.06.642, chXHA.06.275,he.06.642-1, he.06.642-2, he.06.275-1, he.06.275-2, he.06.275-3,he.06.275-4, XPA.06.128, XPA.06.129, XPA.06.130, XPA.06.131, XPA.06.141,XPA.06.147, XPA.06.148, XPA.06.158, XPA.06.159, XPA.06.163, XPA.06.167,XPA.06.171, XPA.06.178, XPA.06.181, XPA.06.192, XPA.06.202, XPA.06.203,XPA.06.206, XPA.06.207, XPA.06.210, XPA.06.212, XPA.06.217, XPA.06.219,XPA.06.229, XPA.06.233, XPA.06.235, XPA.06.239, XPA.06.145, XHA.06.567,XHA.06.642, XHA.06.983, XHA.06.275, XHA.06.189, or XHA.06.907.

In another embodiment, an aforementioned antibody comprises a constantregion of a human antibody sequence and one or more heavy and lightchain variable framework regions of a human antibody sequence. In yetanother embodiment of the invention, an aforementioned antibody isprovided wherein the human antibody sequence is an individual humansequence, a human consensus sequence, an individual human germlinesequence, or a human consensus germline sequence.

In still another embodiment, an aforementioned antibody is providedwherein the heavy chain constant region is a modified or unmodified IgG,IgM, IgA, IgD, IgE, a fragment thereof, or combinations thereof. Inanother embodiment, an aforementioned antibody is provided wherein theheavy chain constant region is a modified or unmodified IgG1, IgG2, IgG3or IgG4. In another embodiment, an aforementioned antibody is providedthat has an equilibrium dissociation constant of 10⁻⁶, 10⁻⁷, 10⁻⁸ or10⁻⁹ M or lower to PRLR. In yet another embodiment, an aforementionedantibody is provided comprising a conservative substitution in the CDRs.In another embodiment, an aforementioned antibody is provided comprisinga conservative or non-conservative change in low and moderate riskresidues. In still another embodiment, an aforementioned antibody isprovided wherein the light chain constant region is a modified orunmodified lambda light chain constant region, a kappa light chainconstant region, a fragment thereof, or combinations thereof.

In yet another embodiment, an aforementioned antibody is provided thatinhibits PRLR dimerization, inhibits PRLR intracellular phosphorylation,inhibits the induction of MAPK phosphorylation, inhibits the inductionof Stat5 phosphorylation, inhibits the induction of AKT phosphorylation,and/or inhibits the binding of PRL to PRLR.

In other embodiments, an aforementioned antibody further inhibits VEGFproduction and/or angiogenesis.

In yet another embodiment, an aforementioned antibody is provided thatinhibits the proliferation of a cancer cell. In yet another embodiment,the antibody inhibits proliferation of a breast, prostate, or lungcancer cell.

In addition to cancer, another embodiment of the invention provides anaforementioned antibody for the prevention and/or treatment ofautoimmune and inflammatory diseases or disorders. The antibodies areespecially useful in preventing, ameloriating, or treating diseasescomprising an autoimmune and/or inflammatory component. These diseasesinclude, but are not limited to, autoimmune and inflammatory diseasessuch as systemic lupus erythematosus (SLE), discoid lupus, lupusnephritis, sarcoidosis, inflammatory arthritis, including, but notlimited to, juvenile arthritis, rheumatoid arthritis, psoriaticarthritis, Reiter's syndrome, ankylosing spondylitis, and goutyarthritis, rejection of an organ or tissue transplant, hyperacute,acute, or chronic rejection and/or graft versus host disease, multiplesclerosis, hyper IgE syndrome, polyarteritis nodosa, primary biliarycirrhosis, inflammatory bowel disease, Crohn's disease, celiac's disease(gluten-sensitive enteropathy), autoimmune hepatitis, pernicious anemia,autoimmune hemolytic anemia, psoriasis, scleroderma, myasthenia gravis,autoimmune thrombocytopenic purpura, autoimmune thyroiditis, Grave'sdisease, Hashimoto's thyroiditis, immune complex disease, chronicfatigue immune dysfunction syndrome (CFIDS), polymyositis anddermatomyositis, cryoglobulinemia, thrombolysis, cardiomyopathy,pemphigus vulgaris, pulmonary interstitial fibrosis, Type I and Type IIdiabetes mellitus, type 1, 2, 3 and 4 delayed-type hypersensitivity,allergy or allergic disorders, unwanted/unintended immune responses totherapeutic proteins, asthma, Churg-Strauss syndrome (allergicgranulomatosis), atopic dermatitis, allergic and irritant contactdermatitis, urtecaria, IgE-mediated allergy, atherosclerosis,vasculitis, idiopathic inflammatory myopathies, hemolytic disease,Alzheimer's disease, chronic inflammatory demyelinating polyneuropathy,and the like.

In another embodiment, an aforementioned antibody is provided that isconjugated to another diagnostic or therapeutic agent.

In still another embodiment, a method of screening for an antibody tothe extracellular domain of a PRLR protein useful for the treatment ofcancer is provided comprising the steps of: contacting a polypeptidecomprising the ECD of PRLR with a candidate antibody that contains atleast 1, 2, 3, 4, 5, or 6 CDRs of antibodies chXHA.06.642, chXHA.06.275,he.06.642-1, he.06.642-2, he.06.275-1, he.06.275-2, he.06.275-3,he.06.275-4, XPA.06.128, XPA.06.129, XPA.06.130, XPA.06.131, XPA.06.141,XPA.06.147, XPA.06.148, XPA.06.158, XPA.06.159, XPA.06.163, XPA.06.167,XPA.06.171, XPA.06.178, XPA.06.181, XPA.06.192, XPA.06.202, XPA.06.203,XPA.06.206, XPA.06.207, XPA.06.210, XPA.06.212, XPA.06.217, XPA.06.219,XPA.06.229, XPA.06.233, XPA.06.235, XPA.06.239, XPA.06.145, XHA.06.567,XHA.06.642, XHA.06.983, XHA.06.275, XHA.06.189, and XHA.06.907;detecting binding affinity of the candidate antibody to the polypeptide,and identifying said candidate antibody as an antibody useful for thetreatment of cancer if an equilibrium dissociation constant of 10⁻⁶ M orlower is detected.

In another embodiment, a method of systematically altering antibodiesand screening for an antibody to the extracellular domain of a PRLRprotein useful for the treatment of cancer is provided comprising thesteps of preparing a candidate antibody that contains modifications toone or two amino acids within the CDRs of antibodies chXHA.06.642,chXHA.06.275, he.06.642-1, he.06.642-2, he.06.275-1, he.06.275-2,he.06.275-3, he.06.275-4, XPA.06.128, XPA.06.129, XPA.06.130,XPA.06.131, XPA.06.141, XPA.06.147, XPA.06.148, XPA.06.158, XPA.06.159,XPA.06.163, XPA.06.167, XPA.06.171, XPA.06.178, XPA.06.181, XPA.06.192,XPA.06.202, XPA.06.203, XPA.06.206, XPA.06.207, XPA.06.210, XPA.06.212,XPA.06.217, XPA.06.219, XPA.06.229, XPA.06.233, XPA.06.235, XPA.06.239,XPA.06.145, XHA.06.567, XHA.06.642, XHA.06.983, XHA.06.275, XHA.06.189,and XHA.06.907; contacting a polypeptide comprising the ECD of PRLR withsaid candidate antibody; detecting binding affinity of the candidateantibody to the polypeptide; and identifying said candidate antibody asan antibody useful for the treatment of cancer if an equilibriumdissociation constant of 10⁻⁶ M or lower is detected.

In still another embodiment, a method of screening for an antibody tothe extracellular domain of a PRLR protein useful for the treatment ofcancer comprising the steps of contacting a breast, lung, or prostatecell with a candidate antibody that contains at least 1, 2, 3, 4, 5 or 6CDRs of antibodies chXHA.06.642, chXHA.06.275, he.06.642-1, he.06.642-2,he.06.275-1, he.06.275-2, he.06.275-3, he.06.275-4, XPA.06.128,XPA.06.129, XPA.06.130, XPA.06.131, XPA.06.141, XPA.06.147, XPA.06.148,XPA.06.158, XPA.06.159, XPA.06.163, XPA.06.167, XPA.06.171, XPA.06.178,XPA.06.181, XPA.06.192, XPA.06.202, XPA.06.203, XPA.06.206, XPA.06.207,XPA.06.210, XPA.06.212, XPA.06.217, XPA.06.219, XPA.06.229, XPA.06.233,XPA.06.235, XPA.06.239, XPA.06.145, XHA.06.567, XHA.06.642, XHA.06.983,XHA.06.275, XHA.06.189, and XHA.06.907 or an antibody that contains amodification of one or two amino acids within one or more CDRs;detecting proliferation or survival of said cell; and identifying saidcandidate antibody as an antibody useful for the treatment of cancer ifa decrease in cell proliferation or survival is detected.

In still another embodiment, a method of treating a subject sufferingfrom cancer, including a subject suffering from stage 0, I, II, III, IVor V cancer, comprising the step of administering an aforementionedantibody in a therapeutically effective amount. In a related embodiment,the cancer is breast, lung or prostate cancer. In another embodiment, asecond therapeutic agent is administered. In an exemplary embodiment,the second therapeutic agent is doxorubicin, daunorubicin, or otheranthracycline or topoisomerase inhibitor. In further embodiments, any ofthe foregoing topoisomerase inhibitors are administered withchXHA.06.642, chXHA.06.275, he.06.642-1, he.06.642-2, he.06.275-1,he.06.275-2, he.06.275-3, he.06.275-4. In still another embodiment, thesubject is further treated with radiation therapy or surgery. In stillanother embodiment of the invention, the subject is positive for PRLRexpression and HER2-neu expression, and wherein said second therapeuticagent is an anti-Her2-neu antibody. In a related embodiment, the subjectis positive for PRLR expression and ER expression, and wherein saidsecond therapeutic agent is an anti-ER antibody. In a furtherembodiment, the invention provides an antibody of the invention for usein medicine, including for use in treating a cancer. In otherembodiments, the invention provides the use of an antibody of theinvention in the manufacture of a medicament for treating a cancer. Themedicament may be administered to a patient in combination with a secondtherapeutic agent, and/or with radiation therapy.

In another embodiment of the invention, a method of targeting a tumorcell expressing PRLR is provided comprising the step of administering anaforementioned antibody conjugated to a radionuclide or other toxin. Inanother embodiment, the subject is a mammal. In still anotherembodiment, the subject is a human.

In still another embodiment, an isolated nucleic acid molecule isprovided comprising a nucleotide sequence that encodes the heavy chainor light chain of an aforementioned antibody. In still anotherembodiment, an expression vector comprising the aforementioned nucleicacid molecule operably linked to a regulatory control sequence isprovided. In yet another embodiment, a host cell comprising theaforementioned vector or the aforementioned nucleic acid molecule isprovided.

In still another embodiment, a method of using the aforementioned hostcell to produce an antibody, comprising culturing the host cell undersuitable conditions and recovering said antibody is provided. In stillanother embodiment, the antibody produced by the aforementioned methodis provided.

In still another embodiment, an aforementioned antibody that is purifiedto at least 95% homogeneity by weight is provided. In anotherembodiment, a pharmaceutical composition comprising the aforementionedantibody and a pharmaceutically acceptable carrier is provided.

In yet another embodiment, a kit comprising an aforementioned antibodycomprising a therapeutically effective amount of an antibody of theinvention, packaged in a container, the kit optionally containing asecond therapeutic agent, and further comprising a label attached to orpackaged with the container, the label describing the contents of thecontainer and providing indications and/or instructions regarding use ofthe contents of the container to treat cancer, is provided. In anotherembodiment, the kit is provided wherein the container is a vial orbottle or prefilled syringe.

In another embodiment of the invention, an antibody that binds theextracellular domain of PRLR comprising a variable light chain aminoacid sequence selected from the group consisting of SEQ ID NO: 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,61, 63, 65, 67, 69, 71, 73, 75, 82, 84, 86, 88, 91, 95 and 96, isprovided. In another embodiment, an antibody that binds theextracellular domain of PRLR is provided comprising a variable heavychain amino acid sequence selected from the group consisting of SEQ IDNO: 20, 2, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 83, 85, 87, 89, 90, 93, 94,97 and 98. In yet another embodiment, an antibody that binds theextracellular domain of PRLR comprising a variable light chain aminoacid sequence SEQ ID NO: 88, and a variable heavy chain amino acidsequence of SEQ ID NO: 89, is provided. In still another embodiment, anantibody that binds the extracellular domain of PRLR comprising avariable light chain amino acid sequence SEQ ID NO: 88, and a variableheavy chain amino acid sequence of SEQ ID NO: 90 is provided.

In yet another embodiment of the invention, an antibody is provided thatbinds the extracellular domain of PRLR comprising a variable light chainamino acid sequence of SEQ ID NO: 91, and a variable heavy chain aminoacid sequence of SEQ ID NO: 93. In another embodiment, an antibody thatbinds the extracellular domain of PRLR comprising a variable light chainamino acid sequence of SEQ ID NO: 91, and a variable heavy chain aminoacid sequence of SEQ ID NO: 94 is provided. In still another embodiment,an antibody that binds the extracellular domain of PRLR comprising avariable light chain amino acid sequence of SEQ ID NO: 92, and avariable heavy chain amino acid sequence of SEQ ID NO: 93. In yetanother embodiment of the invention, an antibody that binds theextracellular domain of PRLR is provided comprising a variable lightchain amino acid sequence of SEQ ID NO: 92, and a variable heavy chainamino acid sequence of SEQ ID NO: 94.

In still another embodiment of the invention, an antibody that binds theextracellular domain of PRLR is provided comprising a variable lightchain amino acid sequence of SEQ ID NO: 95, and a variable heavy chainamino acid sequence of SEQ ID NO: 97. In another embodiment, an antibodythat binds the extracellular domain of PRLR comprising a variable lightchain amino acid sequence of SEQ ID NO: 95, and a variable heavy chainamino acid sequence of SEQ ID NO: 98 is provided. In yet anotherembodiment, an antibody that binds the extracellular domain of PRLRcomprising a variable light chain amino acid sequence of SEQ ID NO: 96,and a variable heavy chain amino acid sequence of SEQ ID NO: 97 isprovided. In another embodiment, an antibody that binds theextracellular domain of PRLR is provided comprising a variable lightchain amino acid sequence of SEQ ID NO: 96, and a variable heavy chainamino acid sequence of SEQ ID NO: 98.

In another embodiment of the invention, an antibody that binds theextracellular domain of human PRLR with a KD of at least 10 to 25,000fold, 100 to 20,000 fold, 1,000 to 18,000 fold, 5,000 to 17,000 fold,8,000 to 16,000 fold, 10,000 to 15,000 fold, 12,000 to 15,000 fold, or13,000 to 14,000 fold, fold lower than the extracellular domain ofmurine PRLR is provided. In a related embodiment, the aforementionedantibody binds the same epitope as he.06.275-4. In still anotherembodiment, an antibody that binds the extracellular domain of humanPRLR, the extracellular domain of murine PRLR, and the extracellulardomain of rat PRLR is provided. In another embodiment, an antibody thatbinds the extracellular domain of human, murine and rat PRLR with anequilibrium dissociation constant (K_(D)) of 10⁻⁶ M or lower isprovided. In a related embodiment, the aforementioned antibody binds thesame epitope as he.06.642-2.

In still another embodiment, the above methods can be used to identify asubject in need of treatment with an anti-PRLR antibody by, for example,(a) obtaining a sample from the subject; and (b) analyzing the samplefor level of phosphorylation of PRLR, Jak2, Mapk, Stat5, Erk1/2 and/orAkt; wherein the level of phosphorylation of PRLR, Jak2, Mapk, Stat5,Erk1/2 and/or Akt is indicative of a need for treatment with ananti-PRLR antibody. In another embodiment, a method of monitoring cancertherapy in a subject afflicted with cancer is provided comprising thesteps of: (a) analyzing a first sample from the subject for level ofphosphorylation of PRLR prior to the initiation of treatment with acancer therapeutic; and (b) analyzing a second sample after theinitiation of the treatment with the cancer therapeutic, wherein areduction in the level of phosphorylated PRLR after the initiation ofthe treatment with the cancer therapeutic indicates the patient isreceiving a therapeutically effective dose of the cancer therapeutic. Ina related embodiment, the cancer therapeutic is an antibody according toany one of the above described embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the gene arrangement and exons in various isoforms of PRLR.

FIGS. 2, 3 and 4 show effect of selected PRLR-specific antibodies onpERK1/2 phosphorylation. [mAb 1167 is a control murine anti-PRLRmonoclonal antibody; R&D Systems, catalog #MAB1167]

FIG. 5 shows the effect of PRLR-specific antibody on proliferation of aPRL-responsive tumor cell line.

FIG. 6 shows the effect of PRLR-specific antibody on PRLR intracellularphosphorylation.

FIG. 7A-7C shows the VH and VL amino acid sequences, as well as thelocation of the CDRs (underlined), of antibodies XPA.06.128, XPA.06.129,XPA.06.130, XPA.06.131, XPA.06.141, XPA.06.147, XPA.06.148, XPA.06.158,XPA.06.159, XPA.06.163, XPA.06.167, XPA.06.171, XPA.06.178, XPA.06.181,XPA.06.192, XPA.06.202, XPA.06.203, XPA.06.206, XPA.06.207, XPA.06.210,XPA.06.212, XPA.06.217, XPA.06.219, XPA.06.229, XPA.06.233, XPA.06.235,XPA.06.239 which had greater than 80% inhibition in the pERK assay.

FIG. 8 shows the VH and VL amino acid sequence of antibody XPA.06.145.

FIG. 9 shows the leader and VH and VL nucleotide sequences of antibodiesXHA.06.983, XHA.06.275, and XHA.06.642.

FIG. 10 shows the VH and VL amino acid sequences of antibodiesXHA.06.983, XHA.06.275, and XHA.06.642 (CDRs underlined).

FIG. 11 shows chimeric anti-PRLR mAbs chXHA.06.642, chXHA.06.275, andchXHA.06.983 potently inhibit the proliferation and survival of BaF/PRLRcells. KLH-G1 is a non-specific isotype matched control antibody. Panelat right shows IC50 values of corresponding mouse mAbs.

FIG. 12 shows chimeric anti-PRLR mAbs inhibit STAT5 signaling in T47Dcells. Cells were pre-treated with 1 ug/ml mAb prior to 30 minstimulation with 50 ng/ml PRL. Lysates were analyzed for the presence ofphospho-PRLR using antibodies specific for phosphotyrosine residues 546and 611 of the PRLR.

FIG. 13 shows the effect of Human Engineered™ antibodies on pERK1/2phosphorylation.

FIG. 14 shows ADCC mediated by chimeric anti-PRLR mAbs. T47D-T2 cellswere labeled with Calcein-AM prior to application of mAb (1 ug/ml) andpurified human NK cells at an effector-to-target ratio of 10:1.Following a 4 hr incubation, Calcein-AM release into the supernatant wasmeasured. Anti-KLH antibody and Herceptin™ were used as negative andpositive controls, respectively. % specific lysis was calculated as(experimental release−spontaneous release)/(maximal release−spontaneousrelease)×100.

FIG. 15 shows Anti-PRLR mAbs synergize with cytotoxic drugs incombination studies. Doxorubicin (top panel) and Cisplatin (bottompanel) were co-administered with anti-KLH control Ab, anti-PRLR mAbchXHA.06.642, or anti-PRLR mAb chXHA.06.275 (all at 1 ug/ml). Cellsurvival was determined by CellTiter Glo™ analysis and is reported asRLU (y-axis).

FIG. 16 shows Human Engineered™ mAb retain anti-PRLR functionalcharacteristics in STAT5 phosphorylation assays. T47D cells wereincubated with 1 or 10 ug/ml mAb, then treated with or without PRL (50ng/ml) for an additional 30 min.

FIGS. 17A and B show humanized anti-PRLR antibody candidates potentlyinhibit the growth of PRL-dependent BaF3/PRLR cells. BaF3/PRLR cellswere grown in the presence of PRL (50 ng/ml) for 48 hr with eitheranti-KLH control antibody (top line), chimeric antibody, or HumanEngineered™ versions. EC50 values were calculated using non-linearregression analysis of the curve fits.

FIGS. 18A and B show inhibition of p-STAT5 in Nb2-C11 tumors ofchXHA.06.642 treated animals. Athymic mice with subcutaneous Nb2-c11tumors were injected intraperitoneally with chXHA.06.642 or KLH controlIgG1 mAb. Two days later a 20 ug bolus intraperitoneal injection of oPRLwas administered. Control animals were injected with saline. Two dayslater a 20 ug bolus injection of oPRL was administeredintraperitoneally, and 40 minutes later tumors were collected andevaluated for p-STAT5 by immunoblot or IHC. FIG. 18A, Western blot of 80ug of Tyr694 p-STAT5; FIG. 18B, IHC of Tyr694 p-STAT5.

FIGS. 19A and B show that chXHA.06.642 is efficacious in the Nb2-C11 ratlymphoma model in SCID mice in two different studies.

FIGS. 20A and B show chXHA.06.642 regresses established Nb2-C11 ratlymphoma tumors in SCID mice. FIG. 20A displays tumor volume; FIG. 20Bdisplays conditional survival.

FIGS. 21A and B show intraperitoneal bolus injection of oPRL inducesp-STAT5, and treatment with chA64.1 inhibits p-STAT5 induction in T47Dhuman breast xenografts. chXHA.06.642 or KLH control IgG1 were injectedintraperitoneally into T47D tumor bearing immunocompromised miceimplanted with 0.18 mg/day estradiol (E₂) pellets to support growth. Twodays later a 20 ug bolus injection of oPRL was administeredintraperitoneally, and 40 minutes later T47D tumors were collected andevaluated for p-STAT5 by immunoblot or IHC. p-ERK or p-AKT levels werealso evaluated by immunoblot. FIG. 21A, Western blot of 80 ug of Tyr694p-STAT5; FIG. 21B, IHC of Tyr694 p-STAT5.

DETAILED DESCRIPTION

The present invention provides PRLR-specific antibodies, pharmaceuticalformulations containing such antibodies, methods of preparing theantibodies and pharmaceutical formulations, and methods of treatingpatients with the pharmaceutical formulations and compounds. Antibodiesaccording to the present invention may have a desired biologicalactivity of binding to PRLR and/or inhibiting the dimerization of PRLRand/or inhibiting PRLR intracellular phosphorylation, and/or inhibitingPRLR downstream signaling, e.g. through phosphorylation of Jak2, Mapk,Stat5, Erk1/2 and/or Akt, and inhibiting cellular proliferationassociated with cancer or tumors. In this way, direct analysis of PRLRactivation by detection of its phosphorylation or by assessing thephosphorylation status of other downstream signaling partners such asJak2, Stat5, Erk1/2 and/or Akt, is contemplated. Analysis of downstreamsignaling pathways may thus be used to identify patients in need ofanti-PRLR antibodies or used to monitor patients who have been treatedwith anti-PRLR antibodies.

Antibodies according to the present invention may alternatively (or inaddition) have a desired biological activity of binding to PRLRexpressed on cancer cells, thus serving to target cytotoxic therapies tothe cancer cells.

The invention further relates to screening assays to identifyantagonists or agonists of a PRLR gene or gene product and variantsthereof. Thus, the invention relates to methods for identifying agonistsor antagonists of a PRLR gene or gene product and variants thereof, andthe use of said agonist or antagonist to treat or prevent cancer asdescribed herein. Additionally, the present invention contemplates useof the nucleic acid molecules, polypeptides, and/or antagonists oragonists of gene products encoded a PRLR gene to screen, diagnose,prevent and/or treat disorders characterized by aberrant expression oractivity of PRLR, which include, cancers, such as but not limited tocancer of the lung, breast, and prostate.

Several preferred murine or chimeric antibodies with high affinity andpotency as measured by in vitro assays are modified to be lessimmunogenic in humans based on the Human Engineering™ method ofStudnicka et al. Briefly, surface exposed amino acid residues of theheavy chain and light chain variable regions are modified to humanresidues in positions determined to be unlikely to adversely effecteither antigen binding or protein folding, while reducing itsimmunogenicity with respect to a human environment. Synthetic genescontaining modified heavy and/or light chain variable regions areconstructed and linked to human y heavy chain and/or kappa light chainconstant regions. Any human heavy chain and light chain constant regionsmay be used in combination with the Human Engineered™ antibody variableregions. The human heavy and light chain genes are introduced intomammalian cells and the resultant recombinant immunoglobulin productsare obtained and characterized.

Exemplary antibodies according to the invention include chXHA.06.642,chXHA.06.275, he.06.642-1, he.06.642-2, he.06.275-1, he.06.275-2,he.06.275-3, he.06.275-4, XPA.06.128, XPA.06.129, XPA.06.130,XPA.06.131, XPA.06.141, XPA.06.147, XPA.06.148, XPA.06.158, XPA.06.159,XPA.06.163, XPA.06.167, XPA.06.171, XPA.06.178, XPA.06.181, XPA.06.192,XPA.06.202, XPA.06.203, XPA.06.206, XPA.06.207, XPA.06.210, XPA.06.212,XPA.06.217, XPA.06.219, XPA.06.229, XPA.06.233, XPA.06.235, XPA.06.239,XPA.06.145, XHA.06.567, XHA.06.642, XHA.06.983, XHA.06.275, XHA.06.189,and XHA.06.907. The following antibody-secreting hybridomas weredeposited with the American Type Culture Collection (ATCC), 10801University Blvd., Manassas, Va. 20110-2209 (USA), pursuant to theprovisions of the Budapest Treaty, on Aug. 17, 2006:

HYBRIDOMA NAME ATCC DEPOSIT NUMBER XHA.06.567 PTA-7794 XHA.06.642PTA-7795 XHA.06.983 PTA-7796 XHA.06.275 PTA-7797 XHA.06.189 PTA-7798XHA.06.907 PTA-7799

The definitions below are provided as an aid to understanding theinvention more completely.

General Definitions

The target antigen human “PRLR” as used herein refers to a humanpolypeptide having substantially the same amino acid sequence as SEQ IDNO: 2 and naturally occurring allelic and/or splice variants thereof.“ECD of PRLR” as used herein refers to the extracellular portion of PRLRrepresented by amino acids 25 to 234 of SEQ ID NO: 2.

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto breast cancer, colon cancer, kidney cancer, liver cancer, lungcancer, lymphoid cancer, ovary cancer, pancreas cancer, prostate cancer,uterine cancer, cervix cancer or skin cancer.

“Treatment” is an intervention performed with the intention ofpreventing the development or altering the pathology of a disorder.Accordingly, “treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Those in need of treatmentinclude those already with the disorder as well as those in which thedisorder is to be prevented. In tumor (e.g., cancer) treatment, atherapeutic agent may directly decrease the pathology of tumor cells, orrender the tumor cells more susceptible to treatment by othertherapeutic agents, e.g., radiation and/or chemotherapy. Treatment ofpatients suffering from clinical, biochemical, radiological orsubjective symptoms of the disease may include alleviating some or allof such symptoms or reducing the predisposition to the disease. The“pathology” of cancer includes all phenomena that compromise the wellbeing of the patient. This includes, without limitation, abnormal oruncontrollable cell growth, metastasis, interference with the normalfunctioning of neighboring cells, release of cytokines or othersecretory products at abnormal levels, suppression or aggravation ofinflammatory or immunological response, etc. Thus, improvement aftertreatment may be manifested as decreased tumor size, decline in tumorgrowth rate, destruction of existing tumor cells or metastatic cells,and/or a reduction in the size or number of metastases.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human.

As used herein, the phrase “therapeutically effective amount” is meantto refer to an amount of therapeutic or prophylactic antibody that wouldbe appropriate for an embodiment of the present invention, that willelicit the desired therapeutic or prophylactic effect or response,including alleviating some or all of such symptoms of disease orreducing the predisposition to the disease, when administered inaccordance with the desired treatment regimen.

Antibodies

“Affinity” or “binding affinity” are often measured by equilibriumassociation constant (K_(A)) or equilibrium dissociation constant(K_(D)). The term “immunospecific” or “specifically binding” means thatthe antibody binds to PRLR or its ECD with an equilibrium associationconstant (K_(A)) of greater than or equal to about 10⁶M⁻¹, greater thanor equal to about 10⁷M⁻¹, greater than or equal to about 10⁸M⁻¹, orgreater than or equal to about 10⁹M⁻¹, 10¹⁰M⁻¹, 10¹¹M⁻¹ or 10¹²M⁻¹. Theantibody may have substantially greater affinity for the target antigencompared to other unrelated molecules. The antibody may also havesubstantially greater affinity for the target antigen compared toorthologs or homologs, e.g. at least 1.5-fold, 2-fold, 5-fold 10-fold,100-fold, 10³-fold, 10⁴-fold, 10⁵-fold, 10⁶-fold or greater relativeaffinity for the target antigen. Alternatively, it might be useful forthe antibody to cross react with a known homolog or ortholog.

Antibodies of the invention may also be characterized by an equilibriumdissociation constant (K_(D)) 10⁻⁴ M, 10⁻⁶ M to 10⁻⁷ M, or 10⁻⁸ M,10⁻¹⁰M, 10⁻¹¹M or 10⁻¹²M or lower. Such affinities may be readilydetermined using conventional techniques, such as by equilibriumdialysis; by using the BIAcore 2000 instrument, using general proceduresoutlined by the manufacturer; by radioimmunoassay using radiolabeledtarget antigen; or by another method known to the skilled artisan. Theaffinity data may be analyzed, for example, by the method of Scatchardet al., Ann N.Y. Acad. Sci., 51:660 (1949).

By “neutralizing antibody” is meant an antibody molecule that is able toeliminate or significantly reduce an effecter function of a targetantigen to which is binds. Accordingly, a “neutralizing” anti-targetantibody is capable of eliminating or significantly reducing an effecterfunction, such as enzyme activity, ligand binding, or intracellularsignaling.

The term “antibody” is used in the broadest sense and includes fullyassembled antibodies, monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies), antibodyfragments that can bind antigen (e.g., Fab′, F′(ab)₂, Fv, single chainantibodies, diabodies), camel bodies and recombinant peptides comprisingthe forgoing as long as they exhibit the desired biological activity.Antibody fragments may be produced by recombinant DNA techniques or byenzymatic or chemical cleavage of intact antibodies and are describedfurther below. Nonlimiting examples of monoclonal antibodies includemurine, chimeric, humanized, human, and Human Engineered™immunoglobulins, antibodies, chimeric fusion proteins having sequencesderived from immunoglobulins, or muteins or derivatives thereof, eachdescribed further below. Multimers or aggregates of intact moleculesand/or fragments, including chemically derivatized antibodies, arecontemplated. Antibodies of any isotype class or subclass arecontemplated according to the present invention.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations that are typicallyinclude different antibodies directed against different determinants(epitopes), each monoclonal antibody is directed against a singledeterminant on the antigen. In addition to their specificity, themonoclonal antibodies are advantageous in that they are synthesized bythe homogeneous culture, uncontaminated by other immunoglobulins withdifferent specificities and characteristics.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler et al., Nature,256:495 [1975], or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). The “monoclonal antibodies” may also berecombinant, chimeric, humanized, human, Human Engineered™, or antibodyfragments, for example.

An “isolated” antibody is one that has been identified and separated andrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would interferewith diagnostic or therapeutic uses for the antibody, and may includeenzymes, hormones, and other proteinaceous or nonproteinaceous solutes.In preferred embodiments, the antibody will be purified (1) to greaterthan 95% by weight of antibody as determined by the Lowry method, andmost preferably more than 99% by weight, (2) to a degree sufficient toobtain at least 15 residues of N-terminal or internal amino acidsequence by use of a spinning cup sequenator, or (3) to homogeneity bySDS-PAGE under reducing or nonreducing conditions using Coomassie blueor, preferably, silver stain. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

An “immunoglobulin” or “native antibody” is a tetrameric glycoprotein.In a naturally-occurring immunoglobulin, each tetramer is composed oftwo identical pairs of polypeptide chains, each pair having one “light”(about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a “variable” region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function.Immunoglobulins can be assigned to different classes depending on theamino acid sequence of the constant domain of their heavy chains. Heavychains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), andepsilon (ε), and define the antibody's isotype as IgM, IgD, IgG, IgA,and IgE, respectively. Several of these may be further divided intosubclasses or isotypes, e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.Different isotypes have different effector functions; for example, IgG1and IgG3 isotypes have ADCC activity. Human light chains are classifiedas kappa (κ) and lambda (λ) light chains. Within light and heavy chains,the variable and constant regions are joined by a “J” region of about 12or more amino acids, with the heavy chain also including a “D” region ofabout 10 more amino acids. See generally, Fundamental Immunology, Ch. 7(Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)).

For a detailed description of the structure and generation ofantibodies, see Roth, D. B., and Craig, N. L., Cell, 94:411-414 (1998),herein incorporated by reference in its entirety. Briefly, the processfor generating DNA encoding the heavy and light chain immunoglobulingenes occurs primarily in developing B-cells. Prior to the rearrangingand joining of various immunoglobulin gene segments, the V, D, J andconstant (C) gene segments are found generally in relatively closeproximity on a single chromosome. During B-cell-differentiation, one ofeach of the appropriate family members of the V, D, J (or only V and Jin the case of light chain genes) gene segments are recombined to formfunctionally rearranged heavy and light immunoglobulin genes. This genesegment rearrangement process appears to be sequential. First, heavychain D-to-J joints are made, followed by heavy chain V-to-DJ joints andlight chain V-to-J joints. In addition to the rearrangement of V, D andJ segments, further diversity is generated in the primary repertoire ofimmunoglobulin heavy and light chain by way of variable recombination atthe locations where the V and J segments in the light chain are joinedand where the D and J segments of the heavy chain are joined. Suchvariation in the light chain typically occurs within the last codon ofthe V gene segment and the first codon of the J segment. Similarimprecision in joining occurs on the heavy chain chromosome between theD and J_(H) segments and may extend over as many as 10 nucleotides.Furthermore, several nucleotides may be inserted between the D and J_(H)and between the V_(H) and D gene segments which are not encoded bygenomic DNA. The addition of these nucleotides is known as N-regiondiversity. The net effect of such rearrangements in the variable regiongene segments and the variable recombination which may occur during suchjoining is the production of a primary antibody repertoire.

“Antibody fragments” comprise a portion of an intact full lengthantibody (including, e.g., human antibodies), preferably the antigenbinding or variable region of the intact antibody, and includemultispecific antibodies formed from antibody fragments. Nonlimitingexamples of antibody fragments include Fab, Fab′, F(ab′)2, Fv, domainantibody (dAb), complementarity determining region (CDR) fragments,single-chain antibodies (scFv), single chain antibody fragments,diabodies, triabodies, tetrabodies, minibodies, linear antibodies(Zapata et al., Protein Eng., 8(10):1057-1062 (1995)); chelatingrecombinant antibodies, tribodies or bibodies, intrabodies, nanobodies,small modular immunopharmaceuticals (SMIPs), an antigen-binding-domainimmunoglobulin fusion protein, a camelized antibody, a VHH containingantibody, or muteins or derivatives thereof, and polypeptides thatcontain at least a portion of an immunoglobulin that is sufficient toconfer specific antigen binding to the polypeptide, such as a CDRsequence, as long as the antibody retains the desired biologicalactivity.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize 35 readily. Pepsin treatment yields an F(ab′)2 fragment thathas two “Fv” fragments. An “Fv” fragment is the minimum antibodyfragment that contains a complete antigen recognition and binding site.This region consists of a dimer of one heavy- and one light-chainvariable domain in tight, non-covalent association. It is in thisconfiguration that the three CDRs of each variable domain interact todefine an antigen binding site on the surface of the VH VL dimer.Collectively, the six CDRs confer antigen-binding specificity to theantibody. However, even a single variable domain (or half of an Fvcomprising only three CDRs specific for an antigen) has the ability torecognize and bind antigen.

“Single-chain Fv” or “sFv” or “scFv” antibody fragments comprise the VHand VL domains of antibody, wherein these domains are present in asingle polypeptide chain. Preferably, the Fv polypeptide furthercomprises a polypeptide linker between the VH and VL domains thatenables the Fv to form the desired structure for antigen binding. For areview of sFv see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994).

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab fragmentsdiffer from Fab′ fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)2 antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem.

The term “hypervariable” region refers to the amino acid residues of anantibody which are responsible for antigen-binding. The hypervariableregion comprises amino acid residues from a “complementarity determiningregion” or CDR [i.e., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) inthe light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102(H3) in the heavy chain variable domain as described by Kabat et al.,Sequences of Proteins of immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)] and/orthose residues from a hypervariable loop (i.e., residues 26-32 (L1),50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32(H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain asdescribed by [Chothia et al., J. Mol. Biol. 196: 901-917 (1987)].

“Framework” or FR residues are those variable domain residues other thanthe hypervariable region residues.

The phrase “constant region” refers to the portion of the antibodymolecule that confers effector functions.

The phrase “chimeric antibody,” as used herein, refers to an antibodycontaining sequence derived from two different antibodies (see, e.g.,U.S. Pat. No. 4,816,567) which typically originate from differentspecies. Most typically, chimeric antibodies comprise human and murineantibody fragments, generally human constant and mouse variable regions.

The term “mutein” or “variant” can be used interchangeably and refers tothe polypeptide sequence of an antibody that contains at least one aminoacid substitution, deletion, or insertion in the variable region or theportion equivalent to the variable region, provided that the mutein orvariant retains the desired binding affinity or biological activity.Muteins may be substantially homologous or substantially identical tothe parent antibody.

The term “derivative” when used in connection with antibodies of theinvention refers to antibodies covalently modified by such techniques asubiquitination, conjugation to therapeutic or diagnostic agents,labeling (e.g., with radionuclides or various enzymes), covalent polymerattachment such as pegylation (derivatization with polyethylene glycol)and insertion or substitution by chemical synthesis of non-natural aminoacids. Derivatives of the invention will retain the binding propertiesof underivatized molecules of the invention.

When used herein, the term “antibody” specifically includes any one ofthe following that retain the ability to bind the extracellular portionof PRLR:

1) an amino acid mutein of a parent antibody having the amino acidsequence set out in FIG. 7A-7C or FIG. 8, including muteins comprising avariable heavy chain amino acid sequence which is at least 60, 65, 70,75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% homologous to theparent amino acid sequence, and/or comprising a variable light chainamino acid sequence which is at least 60, 65, 70, 75, 80, 85, 90, 91,92, 93, 94, 95, 96, 97, 98, or 99% homologous to the parent amino acidsequence, taking into account similar amino acids for the homologydetermination;

2) PRLR-binding polypeptides comprising one or more complementarydetermining regions (CDRs) of a parent antibody having the amino acidsequence set out in FIG. 7A-7C or FIG. 8, preferably comprising at leastCDR3 of the heavy chain, and preferably comprising two or more, or threeor more, or four or more, or five or more, or all six CDRs;

3) Human Engineered™ antibodies generated by altering the parentsequence according to the methods set forth in Studnicka et al., U.S.Pat. No. 5,766,886 and Example 5 herein, using Kabat numbering toidentify low, moderate and high risk residues; such antibodiescomprising at least one of the following heavy chains and at least oneof the following light chains: (a) a heavy chain in which all of the lowrisk rodent residues that differ from corresponding residues in a humanreference immunoglobulin sequence have been modified to be the same asthe human residue in the human reference immunoglobulin sequence or (b)a heavy chain in which all of the low and moderate risk rodent residueshave been modified, if necessary, to be the same residues as in thehuman reference immunoglobulin sequence, (c) a light chain in which allof the low risk residues have been modified, if necessary, to be thesame residues as a human reference immunoglobulin sequence or (b) alight chain in which all of the low and moderate risk residues have beenmodified, if necessary, to be the same residues as a human referenceimmunoglobulin sequence

4) muteins of the aforementioned antibodies in preceding paragraph (3)comprising a heavy or light chain or heavy or light chain variableregions having at least 60% amino acid sequence identity with theoriginal rodent light chain, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, and mostpreferably at least 95%, including for example, 65%, 70%, 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, and 100% identical;

5) PRLR-binding polypeptides comprising the high risk residues of one ormore CDRs of the rodent antibody, and preferably comprising high riskresidues of two or more, or three or more, or four or more, or five ormore, or all six CDRs, and optionally comprising one or more changes atthe low or moderate risk residues;

for example, comprising one or more changes at a low risk residue andconservative substitutions at a moderate risk residue, or

for example, retaining the moderate and high risk amino acid residuesand comprising one or more changes at a low risk residue,

where changes include insertions, deletions or substitutions and may beconservative substitutions or may cause the engineered antibody to becloser in sequence to a human light chain or heavy chain sequence, ahuman germline light chain or heavy chain sequence, a consensus humanlight chain or heavy chain sequence, or a consensus human germline lightchain or heavy chain sequence. Such contemplated changes may also bedisplayed in sequence format as follows. In a hypothetical sequence ofAKKLVHTPYSFKEDF (SEQ ID NO: 99), where the respective risk allotted toeach residue according to Studnicka et al., U.S. Pat. No. 5,766,886, isHMLHMLHMLHMLHML (H=high, M=medium, L=low), exemplary changes to the lowrisk residues of the hypothetical sequence may be displayed as:AKKLVXTPXSFXEDX (SEQ ID NO: 100) where X is any amino acid, oralternatively where X is a conservative substitution of the originalresidue at that position, and exemplary changes to the low and moderaterisk residues can be displayed similarly, e.g. AYXLYXTYXSYXEYX (SEQ IDNO: 101), where X is any amino acid and Y is a conservative substitutionof the original residue at that position.

The term “competing antibody” includes

1) a non-murine or non-rodent monoclonal antibody that binds to the sameepitope of PRLR as antibody chXHA.06.642, chXHA.06.275, he.06.642-1,he.06.642-2, he.06.275-1, he.06.275-2, he.06.275-3, he.06.275-4,XPA.06.128, XPA.06.129, XPA.06.130, XPA.06.131, XPA.06.141, XPA.06.147,XPA.06.148, XPA.06.158, XPA.06.159, XPA.06.163, XPA.06.167, XPA.06.171,XPA.06.178, XPA.06.181, XPA.06.192, XPA.06.202, XPA.06.203, XPA.06.206,XPA.06.207, XPA.06.210, XPA.06.212, XPA.06.217, XPA.06.219, XPA.06.229,XPA.06.233, XPA.06.235, XPA.06.239, XPA.06.145, XHA.06.567, XHA.06.642,XHA.06.983, XHA.06.275, XHA.06.189, or XHA.06.907, e.g. as determinedthrough X-ray crystallography; and/or

2) a non-murine or non-rodent monoclonal antibody that competes withantibody chXHA.06.642, chXHA.06.275, he.06.642-1, he.06.642-2,he.06.275-1, he.06.275-2, he.06.275-3, he.06.275-4, XPA.06.128,XPA.06.129, XPA.06.130, XPA.06.131, XPA.06.141, XPA.06.147, XPA.06.148,XPA.06.158, XPA.06.159, XPA.06.163, XPA.06.167, XPA.06.171, XPA.06.178,XPA.06.181, XPA.06.192, XPA.06.202, XPA.06.203, XPA.06.206, XPA.06.207,XPA.06.210, XPA.06.212, XPA.06.217, XPA.06.219, XPA.06.229, XPA.06.233,XPA.06.235, XPA.06.239, XPA.06.145, XHA.06.567, XHA.06.642, XHA.06.983,XHA.06.275, XHA.06.189, or XHA.06.907, by more than 75%, more than 80%,or more than 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94% or 95%; alternatively, a non-murine or non-rodent monoclonalantibody that reduces the binding of chXHA.06.642, chXHA.06.275,he.06.642-1, he.06.642-2, he.06.275-1, he.06.275-2, he.06.275-3,he.06.275-4, XPA.06.128, XPA.06.129, XPA.06.130, XPA.06.131, XPA.06.141,XPA.06.147, XPA.06.148, XPA.06.158, XPA.06.159, XPA.06.163, XPA.06.167,XPA.06.171, XPA.06.178, XPA.06.181, XPA.06.192, XPA.06.202, XPA.06.203,XPA.06.206, XPA.06.207, XPA.06.210, XPA.06.212, XPA.06.217, XPA.06.219,XPA.06.229, XPA.06.233, XPA.06.235, XPA.06.239, XPA.06.145, XHA.06.567,XHA.06.642, XHA.06.983, XHA.06.275, XHA.06.189, or XHA.06.907 by atleast 2, 3, 4, 5, 6, 10, 20, 50, 100 fold or greater. In one embodiment,the non-murine or non-rodent monoclonal antibody is in 50-fold molarexcess.

Antibodies of the invention preferably bind to the ECD of PRLR with anequilibrium dissociation constant of 10⁻⁶, 10⁻⁷, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹M, 10⁻¹² M or lower and preferably inhibit PRLR intracellularphosphorylation and activation of downstream PRLR signaling, e.g.through activation of STAT5, MAPK, or AKT.

Optionally, any chimeric, human or humanized antibody publicly disclosedbefore the filing date hereof, or disclosed in an application filedbefore the filing date hereof, is excluded from the scope of theinvention.

“Non-rodent” monoclonal antibody is any antibody, as broadly definedherein, that is not a complete intact rodent monoclonal antibodygenerated by a rodent hybridoma. Thus, non-rodent antibodiesspecifically include, but are not limited to, muteins of rodentantibodies, rodent antibody fragments, linear antibodies, chimericantibodies, humanized antibodies, Human Engineered™ antibodies and humanantibodies, including human antibodies produced from transgenic animalsor via phage display technology. Similarly, non-murine antibodiesinclude but are not limited to muteins of murine antibodies, murineantibody fragments, linear antibodies, chimeric, humanized, HumanEngineered™ and human antibodies.

Target Antigen

The target antigen to be used for production of antibodies may be, e.g.,the extracellular portion of PRLR, or a fragment that retains thedesired epitope, optionally fused to another polypeptide that allows theepitope to be displayed in its native conformation. Alternatively,intact PRLR expressed at the surface of cells can be used to generateantibodies. Such cells can be transformed to express PRLR or may beother naturally occurring cells that express PRLR. Other forms of PRLRpolypeptides useful for generating antibodies will be apparent to thoseskilled in the art.

Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. An improved antibody response may be obtainedby conjugating the relevant antigen to a protein that is immunogenic inthe species to be immunized, e.g., keyhole limpet hemocyanin, serumalbumin, bovine thyroglobulin, or soybean trypsin inhibitor using abifunctional or derivatizing agent, for example, maleimidobenzoylsulfosuccinimide ester (conjugation through cysteine residues),N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinicanhydride or other agents known in the art.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later, the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. At 7-14 days post-boosterinjection, the animals are bled and the serum is assayed for antibodytiter. Animals are boosted until the titer plateaus. Preferably, theanimal is boosted with the conjugate of the same antigen, but conjugatedto a different protein and/or through a different cross-linking reagent.Conjugates also can be made in recombinant cell culture as proteinfusions. Also, aggregating agents such as alum are suitably used toenhance the immune response.

Monoclonal Antibodies

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), or may be made byrecombinant DNA methods.

In the hybridoma method, a mouse or other appropriate host animal, suchas a hamster or macaque monkey, is immunized as herein described toelicit lymphocytes that produce or are capable of producing antibodiesthat will specifically bind to the protein used for immunization.Alternatively, lymphocytes may be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)),or can be fused using electrocell fusion.

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofhuman monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).Exemplary murine myeloma lines include those derived from MOP-21 andM.C.-11 mouse tumors available from the Salk Institute Cell DistributionCenter, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells availablefrom the American Type Culture Collection, Rockville, Md. USA.

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). The binding affinity of the monoclonalantibody can, for example, be determined by Scatchard analysis (Munsonet al., Anal. Biochem., 107:220 (1980)).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal. Themonoclonal antibodies secreted by the subclones are suitably separatedfrom the culture medium, ascites fluid, or serum by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies may be isolated and sequencedfrom the hybridoma cells using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of the monoclonal antibodies).Sequence determination will generally require isolation of at least aportion of the gene or cDNA of interest. Usually this requires cloningthe DNA or, preferably, mRNA (i.e., cDNA) encoding the monoclonalantibodies. Cloning is carried out using standard techniques (see, e.g.,Sambrook et al. (1989) Molecular Cloning: A Laboratory Guide, Vols 1-3,Cold Spring Harbor Press, which is incorporated herein by reference).For example, a cDNA library may be constructed by reverse transcriptionof polyA+ mRNA, preferably membrane-associated mRNA, and the libraryscreened using probes specific for human immunoglobulin polypeptide genesequences. In a preferred embodiment, however, the polymerase chainreaction (PCR) is used to amplify cDNAs (or portions of full-lengthcDNAs) encoding an immunoglobulin gene segment of interest (e.g., alight chain variable segment). The amplified sequences can be readilycloned into any suitable vector, e.g., expression vectors, minigenevectors, or phage display vectors. It will be appreciated that theparticular method of cloning used not critical, so long as it ispossible to determine the sequence of some portion of the immunoglobulinpolypeptide of interest. As used herein, an “isolated” nucleic acidmolecule or “isolated” nucleic acid sequence is a nucleic acid moleculethat is either (1) identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the natural source of the nucleic acid or (2) cloned, amplified,tagged, or otherwise distinguished from background nucleic acids suchthat the sequence of the nucleic acid of interest can be determined, isconsidered isolated. An isolated nucleic acid molecule is other than inthe form or setting in which it is found in nature. Isolated nucleicacid molecules therefore are distinguished from the nucleic acidmolecule as it exists in natural cells. However, an isolated nucleicacid molecule includes a nucleic acid molecule contained in cells thatordinarily express the antibody where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

One source for RNA used for cloning and sequencing is a hybridomaproduced by obtaining a B cell from the transgenic mouse and fusing theB cell to an immortal cell. An advantage of using hybridomas is thatthey can be easily screened, and a hybridoma that produces a humanmonoclonal antibody of interest selected. Alternatively, RNA can beisolated from B cells (or whole spleen) of the immunized animal. Whensources other than hybridomas are used, it may be desirable to screenfor sequences encoding immunoglobulins or immunoglobulin polypeptideswith specific binding characteristics. One method for such screening isthe use of phage display technology. Phage display is described in e.g.,Dower et al., WO 91/17271, McCafferty et al., WO 92/01047, and Caton andKoprowski, Proc. Natl. Acad. Sci. USA, 87:6450-6454 (1990), each ofwhich is incorporated herein by reference. In one embodiment using phagedisplay technology, cDNA from an immunized transgenic mouse (e.g., totalspleen cDNA) is isolated, the polymerase chain reaction is used toamplify a cDNA sequences that encode a portion of an immunoglobulinpolypeptide, e.g., CDR regions, and the amplified sequences are insertedinto a phage vector. cDNAs encoding peptides of interest, e.g., variableregion peptides with desired binding characteristics, are identified bystandard techniques such as panning.

The sequence of the amplified or cloned nucleic acid is then determined.Typically the sequence encoding an entire variable region of theimmunoglobulin polypeptide is determined, however, it will sometimes byadequate to sequence only a portion of a variable region, for example,the CDR-encoding portion. Typically the portion sequenced will be atleast 30 bases in length, more often based coding for at least aboutone-third or at least about one-half of the length of the variableregion will be sequenced.

Sequencing can be carried out on clones isolated from a cDNA library,or, when PCR is used, after subcloning the amplified sequence or bydirect PCR sequencing of the amplified segment. Sequencing is carriedout using standard techniques (see, e.g., Sambrook et al. (1989)Molecular Cloning: A Laboratory Guide, Vols 1-3, Cold Spring HarborPress, and Sanger, F. et al. (1977) Proc. Natl. Acad. Sci. USA 74:5463-5467, which is incorporated herein by reference). By comparing thesequence of the cloned nucleic acid with published sequences of humanimmunoglobulin genes and cDNAs, one of skill will readily be able todetermine, depending on the region sequenced, (i) the germline segmentusage of the hybridoma immunoglobulin polypeptide (including the isotypeof the heavy chain) and (ii) the sequence of the heavy and light chainvariable regions, including sequences resulting from N-region additionand the process of somatic mutation. One source of immunoglobulin genesequence information is the National Center for BiotechnologyInformation, National Library of Medicine, National Institutes ofHealth, Bethesda, Md.

Antibody Fragments

As noted above, antibody fragments comprise a portion of an intact fulllength antibody, preferably an antigen binding or variable region of theintact antibody, and include linear antibodies and multispecificantibodies formed from antibody fragments. Nonlimiting examples ofantibody fragments include Fab, Fab′, F(ab′)2, Fv, Fd, domain antibody(dAb), complementarity determining region (CDR) fragments, single-chainantibodies (scFv), single chain antibody fragments, diabodies,triabodies, tetrabodies, minibodies, linear antibodies, chelatingrecombinant antibodies, tribodies or bibodies, intrabodies, nanobodies,small modular immunopharmaceuticals (SMIPs), an antigen-binding-domainimmunoglobulin fusion protein, a camelized antibody, a VHH containingantibody, or muteins or derivatives thereof, and polypeptides thatcontain at least a portion of an immunoglobulin that is sufficient toconfer specific antigen binding to the polypeptide, such as a CDRsequence, as long as the antibody retains the desired biologicalactivity. Such antigen fragments may be produced by the modification ofwhole antibodies or synthesized de novo using recombinant DNAtechnologies or peptide synthesis.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161; and 30 Hollinger et al., Proc.Natl. Acad. Sci. USA, 90:6444-6448 (1993).

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain, and optionally comprising a polypeptide linkerbetween the V_(H) and V_(L) domains that enables the Fv to form thedesired structure for antigen binding (Bird et al., Science 242:423-426,1988, and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988).An Fd fragment consists of the V_(H) and C_(H)1 domains.

Additional antibody fragment include a domain antibody (dAb) fragment(Ward et al., Nature 341:544-546, 1989) which consists of a V_(H)domain.

“Linear antibodies” comprise a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair of antigen bindingregions. Linear antibodies can be bispecific or monospecific (Zapata etal. Protein Eng. 8:1057-62 (1995)).

A “minibody” consisting of scFv fused to CH₃ via a peptide linker(hingeless) or via an IgG hinge has been described in Olafsen, et al.,Protein Eng Des Sel. 2004 April; 17(4):315-23.

Functional heavy-chain antibodies devoid of light chains are naturallyoccurring in nurse sharks (Greenberg et al., Nature 374:168-73, 1995),wobbegong sharks (Nuttall et al., Mol. Immunol. 38:313-26, 2001) andCamelidae (Hamers-Casterman et al., Nature 363: 446-8, 1993; Nguyen etal., J. Mol. Biol. 275: 413, 1998), such as camels, dromedaries, alpacasand llamas. The antigen-binding site is reduced to a single domain, theVH_(H) domain, in these animals. These antibodies form antigen-bindingregions using only heavy chain variable region, i.e., these functionalantibodies are homodimers of heavy chains only having the structure H₂L₂(referred to as “heavy-chain antibodies” or “HCAbs”). Camelized V_(HH)reportedly recombines with IgG2 and IgG3 constant regions that containhinge, CH2, and CH3 domains and lack a CH1 domain (Hamers-Casterman etal., supra). For example, llama IgG1 is a conventional (H₂L₂) antibodyisotype in which V_(H) recombines with a constant region that containshinge, CH1, CH2 and CH3 domains, whereas the llama IgG2 and IgG3 areheavy chain-only isotypes that lack CH1 domains and that contain nolight chains. Classical V_(H)-only fragments are difficult to produce insoluble form, but improvements in solubility and specific binding can beobtained when framework residues are altered to be more VH_(H)-like.(See, e.g., Reichman, et al.,

J Immunol Methods 1999, 231:25-38.) Camelized V_(HH) domains have beenfound to bind to antigen with high affinity (Desmyter et al., J. Biol.Chem. 276:26285-90, 2001) and possess high stability in solution (Ewertet al., Biochemistry 41:3628-36, 2002). Methods for generatingantibodies having camelized heavy chains are described in, for example,in U.S. Patent Publication Nos. 20050136049 and 20050037421.

Because the variable domain of the heavy-chain antibodies is thesmallest fully functional antigen-binding fragment with a molecular massof only 15 kDa, this entity is referred to as a nanobody(Cortez-Retamozo et al., Cancer Research 64:2853-57, 2004). A nanobodylibrary may be generated from an immunized dromedary as described inConrath et al., (Antimicrob Agents Chemother 45: 2807-12, 2001) or usingrecombinant methods as described in

Intrabodies are single chain antibodies which demonstrate intracellularexpression and can manipulate intracellular protein function (Biocca, etal., EMBO J. 9:101-108, 1990; Colby et al., Proc Natl Acad Sci USA.101:17616-21, 2004). Intrabodies, which comprise cell signal sequenceswhich retain the antibody contruct in intracellular regions, may beproduced as described in Mhashilkar et al (EMBO J. 14:1542-51, 1995) andWheeler et al. (FASEB J. 17:1733-5. 2003). Transbodies arecell-permeable antibodies in which a protein transduction domains (PTD)is fused with single chain variable fragment (scFv) antibodies Heng etal., (Med. Hypotheses. 64:1105-8, 2005).

Further contemplated are antibodies that are SMIPs or binding domainimmunoglobulin fusion proteins specific for target protein. Theseconstructs are single-chain polypeptides comprising antigen bindingdomains fused to immunoglobulin domains necessary to carry out antibodyeffector functions. See e.g., WO03/041600, U.S. Patent publication20030133939 and US Patent Publication 20030118592.

Multivalent Antibodies

In some embodiments, it may be desirable to generate multivalent or evena multispecific (e.g. bispecific, trispecific, etc.) monoclonalantibody. Such antibody may have binding specificities for at least twodifferent epitopes of the target antigen, or alternatively it may bindto two different molecules, e.g. to the target antigen and to a cellsurface protein or receptor. For example, a bispecific antibody mayinclude an arm that binds to the target and another arm that binds to atriggering molecule on a leukocyte such as a T-cell receptor molecule(e.g., CD2 or CD3), or Fc receptors for IgG (FcγR), such as FcγRI(CD64), FcγRII (CD32) and FcγRIII (CD16) so as to focus cellular defensemechanisms to the target-expressing cell. As another example, bispecificantibodies may be used to localize cytotoxic agents to cells whichexpress target antigen. These antibodies possess a target-binding armand an arm which binds the cytotoxic agent (e.g., saporin,anti-interferon-60, vinca alkaloid, ricin A chain, methotrexate orradioactive isotope hapten). Multispecific antibodies can be prepared asfull length antibodies or antibody fragments.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Heteroconjugateantibodies may be made using any convenient cross-linking methods.Suitable cross-linking agents are well known in the art, and aredisclosed in U.S. Pat. No. 4,676,980, along with a number ofcross-linking techniques.

According to another approach for making bispecific antibodies, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the C_(H)3 domain of an antibody constant domain. In thismethod, one or more small amino acid side chains from the interface ofthe first antibody molecule are replaced with larger side chains (e.g.,tyrosine or tryptophan). Compensatory “cavities” of identical or similarsize to the large side chain(s) are created on the interface of thesecond antibody molecule by replacing large amino acid side chains withsmaller ones (e.g., alanine or threonine). This provides a mechanism forincreasing the yield of the heterodimer over other unwanted end-productssuch as homodimers. See WO96/27011 published Sep. 6, 1996.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science 229:81 (1985) describe a procedure wherein intact antibodies areproteolytically cleaved to generate F(ab′)₂ fragments. These fragmentsare reduced in the presence of the dithiol complexing agent sodiumarsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes. Better etal., Science 240: 1041-1043 (1988) disclose secretion of functionalantibody fragments from bacteria (see, e.g., Better et al., Skerra etal. Science 240: 1038-1041 (1988)). For example, Fab′-SH fragments canbe directly recovered from E. coli and chemically coupled to formbispecific antibodies (Carter et al., Bio/Technology 10:163-167 (1992);Shalaby et al., J. Exp. Med. 175:217-225 (1992)).

Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the productionof a fully humanized bispecific antibody F(ab′)₂ molecule. Each Fab′fragment was separately secreted from E. coli and subjected to directedchemical coupling in vitro to form the bispecfic antibody. Thebispecific antibody thus formed was able to bind to cells overexpressingthe HER2 receptor and normal human T cells, as well as trigger the lyticactivity of human cytotoxic lymphocytes against human breast tumortargets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers, e.g. GCN4. (See generally Kostelny et al., J. Immunol.148(5):1547-1553 (1992).) The leucine zipper peptides from the Fos andJun proteins were linked to the Fab′ portions of two differentantibodies by gene fusion. The antibody homodimers were reduced at thehinge region to form monomers and then re-oxidized to form the antibodyheterodimers. This method can also be utilized for the production ofantibody homodimers.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. See, for example, Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

Another strategy for making bispecific antibody fragments by the use ofsingle-chain Fv (sFv) dimers has also been reported. See Gruber et al.,J. Immunol. 152: 5368 (1994).

Alternatively, the bispecific antibody may be a “linear antibody”produced as described in Zapata et al. Protein Eng. 8(10):1057-1062(1995). Briefly, these antibodies comprise a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which form a pair of antigen bindingregions. Linear antibodies can be bispecific or monospecific.

Antibodies with more than two valencies are also contemplated. Forexample, trispecific antibodies can be prepared. (Tutt et al., J.Immunol. 147:60 (1991)).

A “chelating recombinant antibody” is a bispecific antibody thatrecognizes adjacent and non-overlapping epitopes of the target antigen,and is flexible enough to bind to both epitopes simultaneously (Neri etal., J Mol Biol. 246:367-73, 1995).

Production of bispecific Fab-scFv (“bibody”) and trispecificFab-(scFv)(2) (“tribody”) are described in Schoonjans et al. (J Immunol.165:7050-57, 2000) and Willems et al. (J Chromatogr B Analyt TechnolBiomed Life Sci. 786:161-76, 2003). For bibodies or tribodies, a scFvmolecule is fused to one or both of the VL-CL (L) and VH-CH₁ (Fd)chains, e.g., to produce a tribody two scFvs are fused to C-term of Fabwhile in a bibody one scFv is fused to C-term of Fab.

Recombinant Production of Antibodies

Antibodies may be produced by recombinant DNA methodology using one ofthe antibody expression systems well known in the art (See, e.g., Harlowand Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory(1988)).

DNA encoding antibodies of the invention may be placed into expressionvectors, which are then transfected into host cells such as E. colicells, simian COS cells, human embryonic kidney 293 cells (e.g., 293Ecells), Chinese hamster ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis ofmonoclonal antibodies in the recombinant host cells. Recombinantproduction of antibodies is well known in the art. Antibody fragmentshave been derived via proteolytic digestion of intact antibodies (see,e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). However,these fragments can now be produced directly by recombinant host cells.Other techniques for the production of antibody fragments, includingpeptide synthesis and covalent linkage, will be apparent to the skilledpractitioner.

Expression control sequences refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is operably linked when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, operably linkedmeans that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

Cell, cell line, and cell culture are often used interchangeably and allsuch designations herein include progeny. Transformants and transformedcells include the primary subject cell and cultures derived therefromwithout regard for the number of transfers. It is also understood thatall progeny may not be precisely identical in DNA content, due todeliberate or inadvertent mutations. Mutant progeny that have the samefunction or biological activity as screened for in the originallytransformed cell are included. Where distinct designations are intended,it will be clear from the context.

In an alternative embodiment, the amino acid sequence of animmunoglobulin of interest may be determined by direct proteinsequencing. Suitable encoding nucleotide sequences can be designedaccording to a universal codon table.

Amino acid sequence muteins of the desired antibody may be prepared byintroducing appropriate nucleotide changes into the encoding DNA, or bypeptide synthesis. Such muteins include, for example, deletions from,and/or insertions into and/or substitutions of, residues within theamino acid sequences of the antibodies. Any combination of deletion,insertion, and substitution is made to arrive at the final construct,provided that the final construct possesses the desired characteristics.The amino acid changes also may alter post-translational processes ofthe monoclonal, human, humanized, Human Engineered™ or mutein antibody,such as changing the number or position of glycosylation sites.

Nucleic acid molecules encoding amino acid sequence muteins of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence muteins) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared muteinor a non-mutein version of the antibody.

The invention also provides isolated nucleic acid encoding antibodies ofthe invention, optionally operably linked to control sequencesrecognized by a host cell, vectors and host cells comprising the nucleicacids, and recombinant techniques for the production of the antibodies,which may comprise culturing the host cell so that the nucleic acid isexpressed and, optionally, recovering the antibody from the host cellculture or culture medium.

For recombinant production of the antibody, the nucleic acid encoding itis isolated and inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. DNA encoding themonoclonal antibody is readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of theantibody). Many vectors are available. The vector components generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more selective marker genes,an enhancer element, a promoter, and a transcription terminationsequence.

(1) Signal sequence component

The antibody of this invention may be produced recombinantly not onlydirectly, but also as a fusion polypeptide with a heterologouspolypeptide, which is preferably a signal sequence or other polypeptidehaving a specific cleavage site at the N-terminus of the mature proteinor polypeptide. The signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. If prokaryotic host cells do not recognize and process thenative antibody signal sequence, the signal sequence may be substitutedby a signal sequence selected, for example, from the group of thepectate lyase (e.g., pelB) alkaline phosphatase, penicillinase, lpp, orheat-stable enterotoxin II leaders. For yeast secretion the nativesignal sequence may be substituted by, e.g., the yeast invertase leader,α factor leader (including Saccharomyces and Kluyveromyces α-factorleaders), or acid phosphatase leader, the C. albicans glucoamylaseleader, or the signal described in WO90/13646. In mammalian cellexpression, mammalian signal sequences as well as viral secretoryleaders, for example, the herpes simplex gD signal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the antibody.

(2) Origin of Replication Component

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ plasmid origin is suitable foryeast, and various viral origins are useful for cloning vectors inmammalian cells. Generally, the origin of replication component is notneeded for mammalian expression vectors (the SV40 origin may typicallybe used only because it contains the early promoter).

(3) Selective Marker Component

Expression and cloning vectors may contain a selective gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, tetracycline, G418, geneticin, histidinol, ormycophenolic acid (b) complement auxotrophic deficiencies, or (c) supplycritical nutrients not available from complex media, e.g., the geneencoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs methotrexate, neomycin, histidinol, puromycin, mycophenolicacid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody-encoding nucleic acid, such as DHFR, thymidine kinase,metallothionein-I and -II, preferably primate metallothionein genes,adenosine deaminase, ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding antibody of the invention, wild-type DHFR protein, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282: 39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85: 12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.Ura3-deficient yeast strains are complemented by plasmids bearing theura3 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135 (1990). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al, Bio/Technology, 9: 968-975(1991).

(4) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to theantibody-encoding nucleic acid. Promoters suitable for use withprokaryotic hosts include the arabinose (e.g., araB) promoter phoApromoter, β-lactamase and lactose promoter systems, alkalinephosphatase, a tryptophan (trp) promoter system, and hybrid promoterssuch as the tac promoter. However, other known bacterial promoters aresuitable. Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding theantibody of the invention.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase or other glycolyticenzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Antibody transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as Abelson leukemia virus, polyoma virus, fowlpox virus,adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcomavirus, most preferably cytomegalovirus, a retrovirus, hepatitis-B virus,Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., theactin promoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297: 598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the rous sarcoma virus long terminal repeat can be used as the promoter.

(5) Enhancer Element Component

Transcription of a DNA encoding the antibody of this invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, alpha-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature 297: 17-18 (1982) on enhancingelements for activation of eukaryotic promoters. The enhancer may bespliced into the vector at a position 5′ or 3′ to the antibody-encodingsequence, but is preferably located at a site 5′ from the promoter.

(6) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding antibody. One useful transcriptiontermination component is the bovine growth hormone polyadenylationregion. See WO94/11026 and the expression vector disclosed therein.Another is the mouse immunoglobulin light chain transcriptionterminator.

(7) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41 Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastors (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibody arederived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified. A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,tobacco, lemna, and other plant cells can also be utilized as hosts.

However, interest has been greatest in vertebrate cells, and propagationof vertebrate cells in culture (tissue culture) has become routineprocedure. Examples of useful mammalian host cell lines are Chinesehamster ovary cells, including CHOK1 cells (ATCC CCL61), DXB-11, DG-44,and Chinese hamster ovary cells/−DHFR (CHO, Urlaub et al., Proc. Natl.Acad. Sci. USA 77: 4216 (1980)); monkey kidney CV1 line transformed bySV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293cells subcloned for growth in suspension culture, [Graham et al., J. GenVirol. 36: 59 (1977)]; baby hamster kidney cells (BHK, ATCC CCL 10);mouse sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980));monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells(VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); humanliver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCCCCL51); TR1 cells (Mather et al., Annals N.Y. Acad. Sci. 383: 44-68(1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

Host cells are transformed or transfected with the above-describedexpression or cloning vectors for antibody production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. In addition, novel vectors and transfected cell lineswith multiple copies of transcription units separated by a selectivemarker are particularly useful and preferred for the expression ofantibodies.

(8) Culturing the Host Cells

The host cells used to produce the antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58: 44 (1979), Barnes et al., Anal.Biochem. 102: 255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866;4,927,762; 4,560,655; or 5,122,469; WO90103430; WO 87/00195; or U.S.Pat. Re. No. 30,985 may be used as culture media for the host cells. Anyof these media may be supplemented as necessary with hormones and/orother growth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as Gentamycin™ drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

(9) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium, including from microbial cultures. If the antibody is producedintracellularly, as a first step, the particulate debris, either hostcells or lysed fragments, is removed, for example, by centrifugation orultrafiltration. Better et al. Science 240: 1041-1043 (1988); ICSU ShortReports 10: 105 (1990); and Proc. Natl. Acad. Sci. USA 90: 457-461(1993) describe a procedure for isolating antibodies which are secretedto the periplasmic space of E. coli. (See also, [Carter et al.,Bio/Technology 10: 163-167 (1992)].

The antibody composition prepared from microbial or mammalian cells canbe purified using, for example, hydroxylapatite chromatography cation oravian exchange chromatography, and affinity chromatography, withaffinity chromatography being the preferred purification technique. Thesuitability of protein A as an affinity ligand depends on the speciesand isotype of any immunoglobulin Fc domain that is present in theantibody. Protein A can be used to purify antibodies that are based onhuman γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and forhuman γ3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix to whichthe affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Wherethe antibody comprises a C_(H) 3 domain, the Bakerbond ABX™ resin (J. T.Baker, Phillipsburg, N.J.) is useful for purification. Other techniquesfor protein purification such as fractionation on an ion-exchangecolumn, ethanol precipitation, Reverse Phase HPLC, chromatography onsilica, chromatography on heparin SEPHAROSE™ chromatography on an anionor cation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Chimeric Antibodies

A rodent antibody on repeated in vivo administration in man either aloneor as a conjugate will bring about an immune response in the recipientagainst the rodent antibody; the so-called HAMA response (Human AntiMouse Antibody). The HAMA response may limit the effectiveness of thepharmaceutical if repeated dosing is required. The immunogenicity of theantibody may be reduced by chemical modification of the antibody with ahydrophilic polymer such as polyethylene glycol or by using geneticengineering methods to make the antibody structure more human like, e.g.chimeric, humanized, human or Human Engineered™ antibodies. Because suchengineered antibodies are less immunogenic in humans than the parentalmouse monoclonal antibodies, they can be used for the treatment ofhumans with far less risk of anaphylaxis. Thus, these antibodies may bepreferred in therapeutic applications that involve in vivoadministration to a human.

Chimeric monoclonal antibodies, in which the variable Ig domains of amouse monoclonal antibody are fused to human constant Ig domains, can begenerated using standard procedures known in the art (See Morrison, S.L., et al. (1984) Chimeric Human Antibody Molecules; Mouse AntigenBinding Domains with Human Constant Region Domains, Proc. Natl. Acad.Sci. USA 81, 6841-6855; and, Boulianne, G. L., et al, Nature 312,643-646. (1984)). For example, the gene sequences for the variabledomains of the rodent antibody which bind CEA can be substituted for thevariable domains of a human myeloma protein, thus producing arecombinant chimeric antibody. These procedures are detailed in EP194276, EP 0120694, EP 0125023, EP 0171496, EP 0173494 and WO 86/01533.Although some chimeric monoclonal antibodies have proved lessimmunogenic in humans, the mouse variable Ig domains can still lead to asignificant human anti-mouse response.

Humanized Antibodies

Humanized antibodies may be achieved by a variety of methods including,for example: (1) grafting the non-human complementarity determiningregions (CDRs) onto a human framework and constant region (a processreferred to in the art as humanizing through “CDR grafting”), or,alternatively, (2) transplanting the entire non-human variable domains,but “cloaking” them with a human-like surface by replacement of surfaceresidues (a process referred to in the art as “veneering”). In thepresent invention, humanized antibodies will include both “humanized”and “veneered” antibodies. These methods are disclosed in, e.g., Joneset al., Nature 321:522 525 (1986); Morrison et al., Proc. Natl. Acad.Sci., U.S.A., 81:6851 6855 (1984); Morrison and Oi, Adv. Immunol., 44:6592 (1988); Verhoeyer et al., Science 239:1534 1536 (1988); Padlan,Molec. Immun. 28:489 498 (1991); Padlan, Molec. Immunol. 31(3):169 217(1994); and Kettleborough, C. A. et al., Protein Eng. 4(7):773 83 (1991)each of which is incorporated herein by reference.

For example, the gene sequences of the CDRs of the rodent antibody maybe isolated or synthesized and substituted for the correspondingsequence regions of a homologous human antibody gene, producing a humanantibody with the specificity of the original rodent antibody. Theseprocedures are described in EP 023940, WO 90/07861 and WO91/09967.

CDR grafting involves introducing one or more of the six CDRs from themouse heavy and light chain variable Ig domains into the appropriatefour framework regions of human variable Ig domains is also called CDRgrafting. This technique (Riechmann, L., et al., Nature 332, 323(1988)), utilizes the conserved framework regions (FR1-FR4) as ascaffold to support the CDR loops which are the primary contacts withantigen. A disadvantage of CDR grafting, however, is that it can resultin a humanized antibody that has a substantially lower binding affinitythan the original mouse antibody, because amino acids of the frameworkregions can contribute to antigen binding, and because amino acids ofthe CDR loops can influence the association of the two variable Igdomains. To maintain the affinity of the humanized monoclonal antibody,the CDR grafting technique can be improved by choosing human frameworkregions that most closely resemble the framework regions of the originalmouse antibody, and by site-directed mutagenesis of single amino acidswithin the framework or CDRs aided by computer modeling of the antigenbinding site (e.g., Co, M. S., et al. (1994), J. Immunol. 152,2968-2976).

One method of humanizing antibodies comprises aligning the non-humanheavy and light chain sequences to human heavy and light chainsequences, selecting and replacing the non-human framework with a humanframework based on such alignment, molecular modeling to predict theconformation of the humanized sequence and comparing to the conformationof the parent antibody. This process is followed by repeated backmutation of residues in the CDR region which disturb the structure ofthe CDRs until the predicted conformation of the humanized sequencemodel closely approximates the conformation of the non-human CDRs of theparent non-human antibody. Such humanized antibodies may be furtherderivatized to facilitate uptake and clearance, e.g., via Ashwellreceptors (See, e.g., U.S. Pat. Nos. 5,530,101 and 5,585,089 whichpatents are incorporated herein by reference).

A number of humanizations of mouse monoclonal antibodies by rationaldesign have been reported (See, for example, 20020091240 published Jul.11, 2002, WO 92/11018 and U.S. Pat. No., 5,693,762, U.S. Pat. No.5,766,866.

Human Engineered™ antibodies

The phrase “Human Engineered™ antibody” refers to an antibody derivedfrom a non-human antibody, typically a mouse monoclonal antibody.Alternatively, a Human Engineered™ antibody may be derived from achimeric antibody that retains or substantially retains the antigenbinding properties of the parental, non-human, antibody but whichexhibits diminished immunogenicity as compared to the parental antibodywhen administered to humans.

Human Engineering™ of antibody variable domains has been described byStudnicka [See, e.g., Studnicka et al. U.S. Pat. No. 5,766,886;Studnicka et al. Protein Engineering 7: 805-814 (1994)] as a method forreducing immunogenicity while maintaining binding activity of antibodymolecules. According to the method, each variable region amino acid hasbeen assigned a risk of substitution. Amino acid substitutions aredistinguished by one of three risk categories: (1) low risk changes arethose that have the greatest potential for reducing immunogenicity withthe least chance of disrupting antigen binding; (2) moderate riskchanges are those that would further reduce immunogenicity, but have agreater chance of affecting antigen binding or protein folding; (3) highrisk residues are those that are important for binding or formaintaining antibody structure and carry the highest risk that antigenbinding or protein folding will be affected. Due to thethree-dimensional structural role of prolines, modifications at prolinesare generally considered to be at least moderate risk changes, even ifthe position is typically a low risk position.

Variable regions of the light and heavy chains of a rodent antibody areHuman Engineered™ as follows to substitute human amino acids atpositions determined to be unlikely to adversely effect either antigenbinding or protein folding, but likely to reduce immunogenicity in ahuman environment. Amino acid residues that are at “low risk” positionsand that are candidates for modification according to the method areidentified by aligning the amino acid sequences of the rodent variableregions with a human variable region sequence. Any human variable regioncan be used, including an individual VH or VL sequence or a humanconsensus VH or VL sequence or an individual or consensus human germlinesequence. The amino acid residues at any number of the low riskpositions, or at all of the low risk positions, can be changed. Forexample, at each low risk position where the aligned murine and humanamino acid residues differ, an amino acid modification is introducedthat replaces the rodent residue with the human residue. Alternatively,the amino acid residues at all of the low risk positions and at anynumber of the moderate risk positions can be changed. Ideally, toachieve the least immunogenicity all of the low and moderate riskpositions are changed from rodent to human sequence.

Synthetic genes containing modified heavy and/or light chain variableregions are constructed and linked to human y heavy chain and/or kappalight chain constant regions. Any human heavy chain and light chainconstant regions may be used in combination with the Human Engineered™antibody variable regions, including IgA (of any subclass, such as IgA1or IgA2), IgD, IgE, IgG (of any subclass, such as IgG1, IgG2, IgG3, orIgG4), or IgM. The human heavy and light chain genes are introduced intohost cells, such as mammalian cells, and the resultant recombinantimmunoglobulin products are obtained and characterized.

Human Antibodies from Transgenic Animals

Human antibodies to target antigen can also be produced using transgenicanimals that have no endogenous immunoglobulin production and areengineered to contain human immunoglobulin loci. For example, WO98/24893 discloses transgenic animals having a human Ig locus whereinthe animals do not produce functional endogenous immunoglobulins due tothe inactivation of endogenous heavy and light chain loci. WO 91/10741also discloses transgenic non-primate mammalian hosts capable ofmounting an immune response to an immunogen, wherein the antibodies haveprimate constant and/or variable regions, and wherein the endogenousimmunoglobulin encoding loci are substituted or inactivated. WO 96/30498discloses the use of the Cre/Lox system to modify the immunoglobulinlocus in a mammal, such as to replace all or a portion of the constantor variable region to form a modified antibody molecule. WO 94/02602discloses non-human mammalian hosts having inactivated endogenous Igloci and functional human Ig loci. U.S. Pat. No. 5,939,598 disclosesmethods of making transgenic mice in which the mice lack endogenousheavy chains, and express an exogenous immunoglobulin locus comprisingone or more xenogeneic constant regions.

Using a transgenic animal described above, an immune response can beproduced to a selected antigenic molecule, and antibody producing cellscan be removed from the animal and used to produce hybridomas thatsecrete human monoclonal antibodies. Immunization protocols, adjuvants,and the like are known in the art, and are used in immunization of, forexample, a transgenic mouse as described in WO 96/33735. Thispublication discloses monoclonal antibodies against a variety ofantigenic molecules including IL 6, IL 8, TNFa, human CD4, L selectin,gp39, and tetanus toxin. The monoclonal antibodies can be tested for theability to inhibit or neutralize the biological activity orphysiological effect of the corresponding protein. WO 96/33735 disclosesthat monoclonal antibodies against IL-8, derived from immune cells oftransgenic mice immunized with IL-8, blocked IL-8 induced functions ofneutrophils. Human monoclonal antibodies with specificity for theantigen used to immunize transgenic animals are also disclosed in WO96/34096 and U.S. patent application no. 20030194404; and U.S. patentapplication no. 20030031667). See also Jakobovits et al., Proc. Natl.Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258(1993); Bruggermann et al., Year in Immuno., 7:33 (1993); and U.S. Pat.No. 5,591,669, U.S. Pat. No. 5,589,369, U.S. Pat. No. 5,545,807; andU.S. Patent Application No. 20020199213, WO 96/34096 and U.S. patentapplication no. 20030194404; and U.S. patent application no.20030031667.

Additional transgenic animals useful to make monoclonal antibodiesinclude the Medarex HuMAb-MOUSE®, described in U.S. Pat. No. 5,770,429and Fishwild, et al. (Nat. Biotechnol. 14:845-851, 1996), which containsgene sequences from unrearranged human antibody genes that code for theheavy and light chains of human antibodies. Immunization of aHuMAb-MOUSE® enables the production of monoclonal antibodies to thetarget protein.

Also, Ishida et al. (Cloning Stem Cells. 4:91-102, 2002) describes theTransChromo Mouse (TCMOUSE™) which comprises megabase-sized segments ofhuman DNA and which incorporates the entire human immunoglobulin (hIg)loci. The TCMOUSE™ has a fully diverse repertoire of hIgs, including allthe subclasses of IgGs (IgG1-G4). Immunization of the TCMOUSE™ withvarious human antigens produces antibody responses comprising humanantibodies.

U.S. Patent Application No. 20030092125 describes methods for biasingthe immune response of an animal to the desired epitope. Humanantibodies may also be generated by in vitro activated B cells (see U.S.Pat. Nos. 5,567,610 and 5,229,275).

Antibodies from Phage Display Technology

The development of technologies for making repertoires of recombinanthuman antibody genes, and the display of the encoded antibody fragmentson the surface of filamentous bacteriophage, has provided a recombinantmeans for directly making and selecting human antibodies, which also canbe applied to humanized, chimeric, murine or mutein antibodies. Theantibodies produced by phage technology are produced as antigen bindingfragments—usually Fv or Fab fragments—in bacteria and thus lack effectorfunctions. Effector functions can be introduced by one of twostrategies: The fragments can be engineered either into completeantibodies for expression in mammalian cells, or into bispecificantibody fragments with a second binding site capable of triggering aneffector function.

Typically, the Fd fragment (V_(H)-C_(H)1) and light chain (V_(L)-C_(L))of antibodies are separately cloned by PCR and recombined randomly incombinatorial phage display libraries, which can then be selected forbinding to a particular antigen. The Fab fragments are expressed on thephage surface, i.e., physically linked to the genes that encode them.Thus, selection of Fab by antigen binding co-selects for the Fabencoding sequences, which can be amplified subsequently. By severalrounds of antigen binding and re-amplification, a procedure termedpanning, Fab specific for the antigen are enriched and finally isolated.

In 1994, an approach for the humanization of antibodies, called “guidedselection”, was described. Guided selection utilizes the power of thephage display technique for the humanization of mouse monoclonalantibody (See Jespers, L. S., et al., Bio/Technology 12, 899-903(1994)). For this, the Fd fragment of the mouse monoclonal antibody canbe displayed in combination with a human light chain library, and theresulting hybrid Fab library may then be selected with antigen. Themouse Fd fragment thereby provides a template to guide the selection.Subsequently, the selected human light chains are combined with a humanFd fragment library. Selection of the resulting library yields entirelyhuman Fab.

A variety of procedures have been described for deriving humanantibodies from phage-display libraries (See, for example, Hoogenboom etal., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol,222:581-597 (1991); U.S. Pat. Nos. 5,565,332 and 5,573,905; Clackson,T., and Wells, J. A., TIBTECH 12, 173-184 (1994)). In particular, invitro selection and evolution of antibodies derived from phage displaylibraries has become a powerful tool (See Burton, D. R., and Barbas III,C. F., Adv. Immunol. 57, 191-280 (1994); and, Winter, G., et al., Annu.Rev. Immunol. 12, 433-455 (1994); U.S. patent application no.20020004215 and WO92/01047; U.S. patent application no. 20030190317published Oct. 9, 2003 and U.S. Pat. No. 6,054,287; U.S. Pat. No.5,877,293.

Watkins, “Screening of Phage-Expressed Antibody Libraries by CaptureLift,” Methods in Molecular Biology, Antibody Phage Display: Methods andProtocols 178: 187-193, and U.S. patent application no. 200120030044772published Mar. 6, 2003 describe methods for screening phage-expressedantibody libraries or other binding molecules by capture lift, a methodinvolving immobilization of the candidate binding molecules on a solidsupport.

The antibody products may be screened for activity and for suitabilityin the treatment methods of the invention using assays as described inthe section entitled “Screening Methods” herein or using any suitableassays known in the art.

Amino Acid Sequence Muteins

Antibodies of the invention include mutein or variants of a parentantibody wherein the polypeptide sequence of the parent antibody hasbeen altered by at least one amino acid substitution, deletion, orinsertion in the variable region or the portion equivalent to thevariable region, including within the CDRs, provided that the mutein orvariant retains the desired binding affinity or biological activity.Muteins may be substantially homologous or substantially identical tothe parent antibody, e.g. at least 65%, 70%, 75%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% identical or homologous. Identity or homology withrespect to this sequence is defined herein as the percentage of aminoacid residues in the candidate sequence that are identical with theparent sequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. None of N-terminal, C-terminal, or internal extensions,deletions, or insertions into the antibody sequence shall be construedas affecting sequence identity or homology. Thus, sequence identity canbe determined by standard methods that are commonly used to compare thesimilarity in position of the amino acids of two polypeptides. Using acomputer program such as BLAST or FASTA, two polypeptides are alignedfor optimal matching of their respective amino acids (either along thefull length of one or both sequences, or along a pre-determined portionof one or both sequences). The programs provide a default openingpenalty and a default gap penalty, and a scoring matrix such as PAM 250[a standard scoring matrix; see Dayhoff et al., in Atlas of ProteinSequence and Structure, vol. 5, supp. 3 (1978)] can be used inconjunction with the computer program. For example, the percent identitycan then be calculated as: the total number of identical matchesmultiplied by 100 and then divided by the sum of the length of thelonger sequence within the matched span and the number of gapsintroduced into the longer sequences in order to align the twosequences.

Antibodies of the invention may also include alterations in thepolypeptide sequence of the constant region, which will not affectbinding affinity but may alter effector function, such asantibody-dependent cellular toxicity (ADCC), complement dependentcytotoxicity (CDC) or clearance and uptake (and resultant effect onhalf-life).

Insertions

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intra-sequence insertions of singleor multiple amino acid residues, e.g. 2, 3 or more. Examples of terminalinsertions include an antibody with an N-terminal methionyl residue orthe antibody (including antibody fragment) fused to an epitope tag or asalvage receptor epitope. Other insertional muteins of the antibodymolecule include the addition of glycosylation sites, addition ofcysteines for intramolecular or intermolecular bonding, or fusion to apolypeptide which increases the serum half-life of the antibody, e.g. atthe N-terminus or C-terminus. For example, cysteine bond(s) may be addedto the antibody to improve its stability (particularly where theantibody is an antibody fragment such as an Fv fragment).

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain. Thepresence of either of these tripeptide sequences in a polypeptidecreates a potential glycosylation site. Thus, N-linked glycosylationsites may be added to an antibody by altering the amino acid sequencesuch that it contains one or more of these tripeptide sequences.O-linked glycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. O-linked glycosylation sites may beadded to an antibody by inserting or substituting one or more serine orthreonine residues to the sequence of the original antibody.

The term “epitope tagged” refers to the antibody fused to an epitopetag. The epitope tag polypeptide has enough residues to provide anepitope against which an antibody there against can be made, yet isshort enough such that it does not interfere with activity of theantibody. The epitope tag preferably is sufficiently unique so that theantibody there against does not substantially cross-react with otherepitopes. Suitable tag polypeptides generally have at least 6 amino acidresidues and usually between about 8-50 amino acid residues (preferablybetween about 9-30 residues). Examples include the flu HA tagpolypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol. 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Mol. Cell. Biol. 5(12): 3610-3616(1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and itsantibody [Paborsky et al., Protein Engineering 3(6): 547-553 (1990)].Other exemplary tags are a poly-histidine sequence, generally around sixhistidine residues, that permits isolation of a compound so labeledusing nickel chelation. Other labels and tags, such as the FLAG® tag(Eastman Kodak, Rochester, N.Y.), well known and routinely used in theart, are embraced by the invention.

As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, orIgG₄) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

Deletions

Amino acid sequence deletions include amino- and/or carboxyl-terminaldeletions ranging in length from one to a hundred or more residues,resulting in fragments that retain binding affinity for target antigen,as well as intra-sequence deletions of single or multiple amino acidresidues, e.g. 2, 3 or more. For example, glycosylation sites may bedeleted or moved to a different position by deleting part or all of thetripeptide or other recognition sequences for glycosylation.

Substitutions

Another type of mutein is an amino acid substitution mutein. Thesemuteins have at least one amino acid residue in the antibody moleculeremoved and a different residue inserted in its place. Substitutionalmutagenesis within any of the hypervariable or CDR regions or frameworkregions is contemplated. Conservative substitutions are shown inTable 1. The most conservative substitution is found under the headingof “preferred substitutions”. If such substitutions result in no changein biological activity, then more substantial changes, denominated“exemplary substitutions” in Table 1, or as further described below inreference to amino acid classes, may be introduced and the productsscreened.

TABLE 1 Preferred Residue Original Exemplary Substitutions Ala (A) val;leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his; asp, lys; glnarg Asp (D) glu; asn glu Cys (C) ser; ala ser Gln (Q) asn; glu asn Glu(E) asp; gln asp Gly (G) ala His (H) asn; gln; lys; arg Ile (I) leu;val; met; ala; leu phe; norleucine Leu (L) norleucine; ile; val; ilemet; ala; phe Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe(F) leu; val; ile; ala; tyr Pro (P) ala Ser (S) thr Thr (T) ser ser Trp(W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met;phe; leu ala; norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side-chainproperties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Conservative substitutions involve replacing an amino acid with anothermember of its class. Non-conservative substitutions involve replacing amember of one of these classes with a member of another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking.

Affinity maturation generally involves preparing and screening antibodyvariants that have substitutions within the CDRs of a parent antibodyand selecting variants that have improved biological properties such asbinding affinity relative to the parent antibody. A convenient way forgenerating such substitutional variants is affinity maturation usingphage display. Briefly, several hypervariable region sites (e.g. 6-7sites) are mutated to generate all possible amino substitutions at eachsite. The antibody variants thus generated are displayed in a monovalentfashion from filamentous phage particles as fusions to the gene IIIproduct of M13 packaged within each particle. The phage-displayedvariants are then screened for their biological activity (e.g. bindingaffinity). See e.g., WO 92/01047, WO 93/112366, WO 95/15388 and WO93/19172.

Current antibody affinity maturation methods belong to two mutagenesiscategories: stochastic and nonstochastic. Error prone PCR, mutatorbacterial strains (Low et al., J. Mol. Biol. 260, 359-68, 1996), andsaturation mutagenesis (Nishimiya et al., J. Biol. Chem. 275:12813-20,2000; Chowdhury, P. S. Methods Mol. Biol. 178, 269-85, 2002) are typicalexamples of stochastic mutagenesis methods (Rajpal et al., Proc NatlAcad Sci USA. 102:8466-71, 2005). Nonstochastic techniques often usealanine-scanning or site-directed mutagenesis to generate limitedcollections of specific variants. Some methods are described in furtherdetail below.

Affinity maturation via panning methods—Affinity maturation ofrecombinant antibodies is commonly performed through several rounds ofpanning of candidate antibodies in the presence of decreasing amounts ofantigen. Decreasing the amount of antigen per round selects theantibodies with the highest affinity to the antigen thereby yieldingantibodies of high affinity from a large pool of starting material.Affinity maturation via panning is well known in the art and isdescribed, for example, in Huls et al. (Cancer Immunol Immunother.50:163-71, 2001). Methods of affinity maturation using phage displaytechnologies are described elsewhere herein and known in the art (seee.g., Daugherty et al., Proc Natl Acad Sci USA. 97:2029-34, 2000).

Look-through mutagenesis—Look-through mutagenesis (LTM) (Rajpal et al.,Proc Natl Acad Sci USA. 102:8466-71, 2005) provides a method for rapidlymapping the antibody-binding site. For LTM, nine amino acids,representative of the major side-chain chemistries provided by the 20natural amino acids, are selected to dissect the functional side-chaincontributions to binding at every position in all six CDRs of anantibody. LTM generates a positional series of single mutations within aCDR where each “wild type” residue is systematically substituted by oneof nine selected amino acids. Mutated CDRs are combined to generatecombinatorial single-chain variable fragment (scFv) libraries ofincreasing complexity and size without becoming prohibitive to thequantitative display of all variants. After positive selection, cloneswith improved binding are sequenced, and beneficial mutations aremapped.

Error-prone PCR—Error-prone PCR involves the randomization of nucleicacids between different selection rounds. The randomization occurs at alow rate by the intrinsic error rate of the polymerase used but can beenhanced by error-prone PCR (Zaccolo et al., J. Mol. Biol. 285:775-783,1999) using a polymerase having a high intrinsic error rate duringtranscription (Hawkins et al., J Mol Biol. 226:889-96, 1992). After themutation cycles, clones with improved affinity for the antigen areselected using routine methods in the art.

DNA Shuffling—Nucleic acid shuffling is a method for in vitro or in vivohomologous recombination of pools of shorter or smaller polynucleotidesto produce variant polynucleotides. DNA shuffling has been described inU.S. Pat. No. 6,605,449, U.S. Pat. No. 6,489,145, WO 02/092780 andStemmer, Proc. Natl. Acad. Sci. USA, 91:10747-51 (1994). Generally, DNAshuffling is comprised of 3 steps: fragmentation of the genes to beshuffled with DNase I, random hybridization of fragments and reassemblyor filling in of the fragmented gene by PCR in the presence of DNApolymerase (sexual PCR), and amplification of reassembled product byconventional PCR.

DNA shuffling differs from error-prone PCR in that it is an inversechain reaction. In error-prone PCR, the number of polymerase start sitesand the number of molecules grows exponentially. In contrast, in nucleicacid reassembly or shuffling of random polynucleotides the number ofstart sites and the number (but not size) of the random polynucleotidesdecreases over time.

In the case of an antibody, DNA shuffling allows the free combinatorialassociation of all of the CDR1s with all of the CDR2s with all of theCDR3s, for example. It is contemplated that multiple families ofsequences can be shuffled in the same reaction. Further, shufflinggenerally conserves the relative order, such that, for example, CDR1will not be found in the position of CDR2. Rare shufflants will containa large number of the best (e.g. highest affinity) CDRs and these rareshufflants may be selected based on their superior affinity.

The template polynucleotide which may be used in DNA shuffling may beDNA or RNA. It may be of various lengths depending on the size of thegene or shorter or smaller polynucleotide to be recombined orreassembled. Preferably, the template polynucleotide is from 50 bp to 50kb. The template polynucleotide often should be double-stranded.

It is contemplated that single-stranded or double-stranded nucleic acidpolynucleotides having regions of identity to the templatepolynucleotide and regions of heterology to the template polynucleotidemay be added to the template polynucleotide, during the initial step ofgene selection. It is also contemplated that two different but relatedpolynucleotide templates can be mixed during the initial step.

Alanine scanning—Alanine scanning mutagenesis can be performed toidentify hypervariable region residues that contribute significantly toantigen binding. Cunningham and Wells, (Science 244:1081-1085, 1989). Aresidue or group of target residues are identified (e.g., chargedresidues such as arg, asp, his, lys, and glu) and replaced by a neutralor negatively charged amino acid (most preferably alanine orpolyalanine) to affect the interaction of the amino acids with antigen.Those amino acid locations demonstrating functional sensitivity to thesubstitutions then are refined by introducing further or other variantsat, or for, the sites of substitution. Thus, while the site forintroducing an amino acid sequence variation is predetermined, thenature of the mutation per se need not be predetermined. For example, toanalyze the performance of a mutation at a given site, ala scanning orrandom mutagenesis is conducted at the target codon or region and theexpressed antibody muteins are screened for the desired activity.

Computer-aided design—Alternatively, or in addition, it may bebeneficial to analyze a crystal structure of the antigen-antibodycomplex to identify contact points between the antibody and antigen, orto use computer software to model such contact points. Such contactresidues and neighboring residues are candidates for substitutionaccording to the techniques elaborated herein. Once such variants aregenerated, the panel of variants is subjected to screening as describedherein and antibodies with superior properties in one or more relevantassays may be selected for further development.

Affinity maturation involves preparing and screening antibody muteinsthat have substitutions within the CDRs of a parent antibody andselecting muteins that have improved biological properties such asbinding affinity relative to the parent antibody. A convenient way forgenerating such substitutional muteins is affinity maturation usingphage display. Briefly, several hypervariable region sites (e.g. 6-7sites) are mutated to generate all possible amino substitutions at eachsite. The antibody muteins thus generated are displayed in a monovalentfashion from filamentous phage particles as fusions to the gene IIIproduct of M13 packaged within each particle. The phage-displayedmuteins are then screened for their biological activity (e.g. bindingaffinity).

Alanine scanning mutagenesis can be performed to identify hypervariableregion residues that contribute significantly to antigen binding.Alternatively, or in addition, it may be beneficial to analyze a crystalstructure of the antigen-antibody complex to identify contact pointsbetween the antibody and antigen. Such contact residues and neighboringresidues are candidates for substitution according to the techniqueselaborated herein. Once such muteins are generated, the panel of muteinsis subjected to screening as described herein and antibodies withsuperior properties in one or more relevant assays may be selected forfurther development.

Altered Effector Function

Other modifications of the antibody are contemplated. For example, itmay be desirable to modify the antibody of the invention with respect toeffector function, so as to enhance the effectiveness of the antibody intreating cancer, for example. For example cysteine residue(s) may beintroduced in the Fc region, thereby allowing interchain disulfide bondformation in this region. The homodimeric antibody thus generated mayhave improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med. 176: 1191-1195 (1992)and Shopes, B. J. Immunol. 148: 2918-2922 (1992). Homodimeric antibodieswith enhanced activity may also be prepared using heterobifunctionalcross-linkers as described in Wolff et al., Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can be engineered which hasdual Fc regions and may thereby have enhanced complement lysis and ADCCcapabilities. See Stevenson et al., Anti-Cancer Drug Design 3: 219-230(1989). In addition, it has been shown that sequences within the CDR cancause an antibody to bind to MHC Class II and trigger an unwanted helperT-cell response. A conservative substitution can allow the antibody toretain binding activity yet lose its ability to trigger an unwantedT-cell response. Also see Steplewski et al., Proc Natl Acad Sci USA.1988; 85(13):4852-6, incorporated herein by reference in its entirety,which described chimeric antibodies wherein a murine variable region wasjoined with human gamma 1, gamma 2, gamma 3, and gamma 4 constantregions.

In certain embodiments of the invention, it may be desirable to use anantibody fragment, rather than an intact antibody, to increase tumorpenetration, for example. In this case, it may be desirable to modifythe antibody fragment in order to increase its serum half-life, forexample, adding molecules such as PEG or other water soluble polymers,including polysaccharide polymers, to antibody fragments to increase thehalf-life. This may also be achieved, for example, by incorporation of asalvage receptor binding epitope into the antibody fragment (e.g., bymutation of the appropriate region in the antibody fragment or byincorporating the epitope into a peptide tag that is then fused to theantibody fragment at either end or in the middle, e.g., by DNA orpeptide synthesis) (see, e.g., WO96/32478).

The salvage receptor binding epitope preferably constitutes a regionwherein any one or more amino acid residues from one or two loops of aFc domain are transferred to an analogous position of the antibodyfragment. Even more preferably, three or more residues from one or twoloops of the Fc domain are transferred. Still more preferred, theepitope is taken from the CH2 domain of the Fc region (e.g., of an IgG)and transferred to the CH1, CH3, or VH region, or more than one suchregion, of the antibody. Alternatively, the epitope is taken from theCH2 domain of the Fc region and transferred to the C.sub.L region orV.sub.L region, or both, of the antibody fragment. See alsoInternational applications WO 97/34631 and WO 96/32478 which describe Fcvariants and their interaction with the salvage receptor.

Thus, antibodies of the invention may comprise a human Fc portion, ahuman consensus Fc portion, or a mutein thereof that retains the abilityto interact with the Fc salvage receptor, including muteins in whichcysteines involved in disulfide bonding are modified or removed, and/orin which the a met is added at the N-terminus and/or one or more of theN-terminal 20 amino acids are removed, and/or regions that interact withcomplement, such as the C1q binding site, are removed, and/or the ADCCsite is removed [see, e.g., Molec. Immunol. 29 (5): 633-9 (1992)].Antibodies of the IgG class may also include a different constantregion, e.g. an IgG2 antibody may be modified to display an IgG1 or IgG4constant region.

In the case of IgG1, modifications to the constant region, particularlythe hinge or CH2 region, may increase or decrease effector function,including ADCC and/or CDC activity. In other embodiments, an IgG2constant region is modified to decrease antibody-antigen aggregateformation. In the case of IgG4, modifications to the constant region,particularly the hinge region, may reduce the formation ofhalf-antibodies. In specific exemplary embodiments, mutating the IgG4hinge sequence Cys-Pro-Ser-Cys to the IgG1 hinge sequenceCys-Pro-Pro-Cys is provided.

Previous studies mapped the binding site on human and murine IgG for FcRprimarily to the lower hinge region composed of IgG residues 233-239.Other studies proposed additional broad segments, e.g. Gly316-Lys338 forhuman Fc receptor I, Lys274-Arg301 and Tyr407-Arg416 for human Fcreceptor III, or found a few specific residues outside the lower hinge,e.g. Asn297 and Glu318 for murine IgG2b interacting with murine Fcreceptor II. The report of the 3.2-Å crystal structure of the human IgG1Fc fragment with human Fc receptor IIIA delineated IgG1 residuesLeu234-Ser239, Asp265-Glu269, Asn297-Thr299, and Ala327-Ile332 asinvolved in binding to Fc receptor IIIA. It has been suggested based oncrystal structure that in addition to the lower hinge (Leu234-Gly237),residues in IgG CH2 domain loops FG (residues 326-330) and BC (residues265-271) might play a role in binding to Fc receptor IIA. See Shields etal., J. Biol. Chem., 276(9):6591-6604 (2001), incorporated by referenceherein in its entirety. Mutation of residues within Fc receptor bindingsites can result in altered effector function, such as altered ADCC orCDC activity, or altered half-life. As described above, potentialmutations include insertion, deletion or substitution of one or moreresidues, including substitution with alanine, a conservativesubstitution, a non-conservative substitution, or replacement with acorresponding amino acid residue at the same position from a differentIgG subclass (e.g. replacing an IgG1 residue with a corresponding IgG2residue at that position).

Shields et al. reported that IgG1 residues involved in binding to allhuman Fc receptors are located in the CH2 domain proximal to the hingeand fall into two categories as follows: 1) positions that may interactdirectly with all FcR include Leu234-Pro238, Ala327, and Pro329 (andpossibly Asp265); 2) positions that influence carbohydrate nature orposition include Asp265 and Asn297. The additional IgG1 residues thataffected binding to Fc receptor II are as follows: (largest effect)Arg255, Thr256, Glu258, Ser267, Asp270, Glu272, Asp280, Arg292, Ser298,and (less effect) His268, Asn276, His285, Asn286, Lys290, Gln295,Arg301, Thr307, Leu309, Asn315, Lys322, Lys326, Pro331, Ser337, Ala339,Ala378, and Lys414. A327Q, A327S, P329A, D265A and D270A reducedbinding. In addition to the residues identified above for all FcR,additional IgG1 residues that reduced binding to Fc receptor IIIA by 40%or more are as follows: Ser239, Ser267 (Gly only), His 268, Glu293,Gln295, Tyr296, Arg301, Val303, Lys338, and Asp376. Muteins thatimproved binding to FcRIIIA include T256A, K290A, S298A, E333A, K334A,and A339T. Lys414 showed a 40% reduction in binding for FcRIIA andFcRIIB, Arg416 a 30% reduction for FcRIIA and FcRIIIA, Gln419 a 30%reduction to FcRIIA and a 40% reduction to FcRIIB, and Lys360 a 23%improvement to FcRIIIA. See also Presta et al., Biochem. Soc. Trans.(2001) 30, 487-490.

For example, U.S. Pat. No. 6,194,551, incorporated herein by referencein its entirety, describes muteins with altered effector functioncontaining mutations in the human IgG Fc region, at amino acid position329, 331 or 322 (using Kabat numbering), some of which display reducedC1q binding or CDC activity. As another example, U.S. Pat. No.6,737,056, incorporated herein by reference in its entirety, describesmuteins with altered effector or Fc-gamma-receptor binding containingmutations in the human IgG Fc region, at amino acid position 238, 239,248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276,278, 280, 283, 285, 286, 289, 290, 292, 294, 295, 296, 298, 301, 303,305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333,334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414,416, 419, 430, 434, 435, 437, 438 or 439 (using Kabat numbering), someof which display receptor binding profiles associated with reduced ADCCor CDC activity. Of these, a mutation at amino acid position 238, 265,269, 270, 327 or 329 are stated to reduce binding to FcRI, a mutation atamino acid position 238, 265, 269, 270, 292, 294, 295, 298, 303, 324,327, 329, 333, 335, 338, 373, 376, 414, 416, 419, 435, 438 or 439 arestated to reduce binding to FcRII, and a mutation at amino acid position238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 293,294, 295, 296, 301, 303, 322, 327, 329, 338, 340, 373, 376, 382, 388,389, 416, 434, 435 or 437 is stated to reduce binding to FcRIII.

U.S. Pat. No. 5,624,821, incorporated by reference herein in itsentirety, reports that C1q binding activity of an murine antibody can bealtered by mutating amino acid residue 318, 320 or 322 of the heavychain and that replacing residue 297 (Asn) results in removal of lyticactivity.

United States Application Publication No. 20040132101, incorporated byreference herein in its entirety, describes muteins with mutations atamino acid positions 240, 244, 245, 247, 262, 263, 266, 299, 313, 325,328, or 332 (using Kabat numbering) or positions 234, 235, 239, 240,241, 243, 244, 245, 247, 262, 263, 264, 265, 266, 267, 269, 296, 297,298, 299, 313, 325, 327, 328, 329, 330, or 332 (using Kabat numbering),of which mutations at positions 234, 235, 239, 240, 241, 243, 244, 245,247, 262, 263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313, 325,327, 328, 329, 330, or 332 may reduce ADCC activity or reduce binding toan Fc gamma receptor.

Chappel et al., Proc Natl Acad Sci USA. 1991; 88(20):9036-40,incorporated herein by reference in its entirety, report that cytophilicactivity of IgG1 is an intrinsic property of its heavy chain CH2 domain.Single point mutations at any of amino acid residues 234-237 of IgG1significantly lowered or abolished its activity. Substitution of all ofIgG1 residues 234-237 (LLGG) into IgG2 and IgG4 were required to restorefull binding activity. An IgG2 antibody containing the entire ELLGGPsequence (residues 233-238) was observed to be more active thanwild-type IgG1.

Isaacs et al., J Immunol. 1998; 161(8):3862-9, incorporated herein byreference in its entirety, report that mutations within a motif criticalfor Fc gammaR binding (glutamate 233 to proline, leucine/phenylalanine234 to valine, and leucine 235 to alanine) completely preventeddepletion of target cells. The mutation glutamate 318 to alanineeliminated effector function of mouse IgG2b and also reduced the potencyof human IgG4.

Armour et al., Mol Immunol. 2003; 40(9):585-93, incorporated byreference herein in its entirety, identified IgG1 muteins which reactwith the activating receptor, FcgammaRIIa, at least 10-fold lessefficiently than wildtype IgG1 but whose binding to the inhibitoryreceptor, FcgammaRIIb, is only four-fold reduced. Mutations were made inthe region of amino acids 233-236 and/or at amino acid positions 327,330 and 331. See also WO 99/58572, incorporated by referehce herein inits entirety.

Xu et al., J Biol. Chem. 1994; 269(5):3469-74, incorporated by referenceherein in its entirety, report that mutating IgG1 Pro331 to Ser markedlydecreased C1q binding and virually eliminated lytic activity. Incontrast, the substitution of Pro for Ser331 in IgG4 bestowed partiallytic activity (40%) to the IgG4 Pro331 mutein.

Schuurman et al., Mol. Immunol. 2001; 38(1):1-8, incorporated byreference herein in its entirety, report that mutating one of the hingecysteines involved in the inter-heavy chain bond formation, Cys226, toserine resulted in a more stable inter-heavy chain linkage. Mutating theIgG4 hinge sequence Cys-Pro-Ser-Cys to the IgG1 hinge sequenceCys-Pro-Pro-Cys also markedly stabilizes the covalent interactionbetween the heavy chains.

Angal et al., Mol. Immunol. 1993; 30(1):105-8, incorporated by referenceherein in its entirety, report that mutating the serine at amino acidposition 241 in IgG4 to proline (found at that position in IgG1 andIgG2) led to the production of a homogeneous antibody, as well asextending serum half-life and improving tissue distribution compared tothe original chimeric IgG4.

The invention also contemplates production of antibody molecules withaltered carbohydrate structure resulting in altered effector activity,including antibody molecules with absent or reduced fucosylation thatexhibit improved ADCC activity. A variety of ways are known in the artto accomplish this. For example, ADCC effector activity is mediated bybinding of the antibody molecule to the FcγRIII receptor, which has beenshown to be dependent on the carbohydrate structure of the N-linkedglycosylation at the Asn-297 of the CH2 domain. Non-fucosylatedantibodies bind this receptor with increased affinity and triggerFcγRIII-mediated effector functions more efficiently than native,fucosylated antibodies. For example, recombinant production ofnon-fucosylated antibody in CHO cells in which the alpha-1,6-fucosyltransferase enzyme has been knocked out results in antibody with100-fold increased ADCC activity [Yamane-Ohnuki et al., BiotechnolBioeng. 2004 Sep. 5; 87(5):614-22]. Similar effects can be accomplishedthrough decreasing the activity of this or other enzymes in thefucosylation pathway, e.g., through siRNA or antisense RNA treatment,engineering cell lines to knockout the enzyme(s), or culturing withselective glycosylation inhibitors [Rothman et al., Mol. Immunol. 1989December; 26(12):1113-23]. Some host cell strains, e.g. Lec13 or rathybridoma YB2/0 cell line naturally produce antibodies with lowerfucosylation levels. Shields et al., J Biol. Chem. 2002 Jul. 26;277(30):26733-40; Shinkawa et al., J Biol. Chem. 2003 Jan. 31;278(5):3466-73. An increase in the level of bisected carbohydrate, e.g.through recombinantly producing antibody in cells that overexpressGnTIII enzyme, has also been determined to increase ADCC activity. Umanaet al., Nat. Biotechnol. 1999 February; 17(2):176-80. It has beenpredicted that the absence of only one of the two fucose residues may besufficient to increase ADCC activity. Ferrara et al., J Biol. Chem. 2005Dec. 5; [Epub ahead of print]

Other Covalent Modifications

Covalent modifications of the antibody are also included within thescope of this invention. They may be made by chemical synthesis or byenzymatic or chemical cleavage of the antibody, if applicable. Othertypes of covalent modifications of the antibody are introduced into themolecule by reacting targeted amino acid residues of the antibody withan organic derivatizing agent that is capable of reacting with selectedside chains or the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,.alpha.-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino-terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing .alpha.-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R-N.dbd.C.dbd.N-R′), where R and R′ aredifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions. Thedeamidated form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the .alpha.-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86(1983)), acetylation of the N-terminal amine, and amidation of anyC-terminal carboxyl group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the antibody. These procedures areadvantageous in that they do not require production of the antibody in ahost cell that has glycosylation capabilities for N- or O-linkedglycosylation. Depending on the coupling mode used, the sugar(s) may beattached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulfhydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan, or (f) the amide group of glutamine. These methods aredescribed in WO87/05330 published 11 Sep. 1987, and in Aplin andWriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

Removal of any carbohydrate moieties present on the antibody may beaccomplished chemically or enzymatically. Chemical deglycosylationrequires exposure of the antibody to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving theantibody intact. Chemical deglycosylation is described by Hakimuddin, etal. Arch. Biochem. Biophys. 259: 52 (1987) and by Edge et al. Anal.Biochem., 118: 131 (1981). Enzymatic cleavage of carbohydrate moietieson antibodies can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al. Meth. Enzymol. 138:350 (1987).

Another type of covalent modification of the antibody comprises linkingthe antibody to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, polyoxyethylated polyols,polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylatedglycerol, polyoxyalkylenes, or polysaccharide polymers such as dextran.Such methods are known in the art, see, e.g. U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192, 4,179,337, 4,766,106,4,179,337, 4,495,285, 4,609,546 or EP 315 456.

Each antibody molecule may be attached to one or more (i.e. 1, 2, 3, 4,5 or more) polymer molecules. Polymer molecules are preferably attachedto antibodies by linker molecules. The polymer may, in general, be asynthetic or naturally occurring polymer, for example an optionallysubstituted straight or branched chain polyalkene, polyalkenylene orpolyoxyalkylene polymer or a branched or unbranched polysaccharide, e.g.homo- or hetero-polysaccharide. Preferred polymers are polyoxyethylenepolyols and polyethylene glycol (PEG). PEG is soluble in water at roomtemperature and has the general formula: R(O—CH2—CH2)n O—R where R canbe hydrogen, or a protective group such as an alkyl or alkanol group.Preferably, the protective group has between 1 and 8 carbons, morepreferably it is methyl. The symbol n is a positive integer, preferablybetween 1 and 1,000, more preferably between 2 and 500. The PEG has apreferred average molecular weight between 1000 and 40,000, morepreferably between 2000 and 20,000, most preferably between 3,000 and12,000. Preferably, PEG has at least one hydroxy group, more preferablyit is a terminal hydroxy group. It is this hydroxy group which ispreferably activated to react with a free amino group on the inhibitor.However, it will be understood that the type and amount of the reactivegroups may be varied to achieve a covalently conjugated PEG/antibody ofthe present invention. Preferred polymers, and methods to attach them topeptides, are shown in U.S. Pat. Nos. 4,766,106; 4,179,337; 4,495,285;and 4,609,546 which are all hereby incorporated by reference in theirentireties.

Gene Therapy

Delivery of a therapeutic antibody to appropriate cells can be effectedvia gene therapy ex vivo, in situ, or in vivo by use of any suitableapproach known in the art, including by use of physical DNA transfermethods (e.g., liposomes or chemical treatments) or by use of viralvectors (e.g., adenovirus, adeno-associated virus, or a retrovirus). Forexample, for in vivo therapy, a nucleic acid encoding the desiredantibody, either alone or in conjunction with a vector, liposome, orprecipitate may be injected directly into the subject, and in someembodiments, may be injected at the site where the expression of theantibody compound is desired. For ex vivo treatment, the subject's cellsare removed, the nucleic acid is introduced into these cells, and themodified cells are returned to the subject either directly or, forexample, encapsulated within porous membranes which are implanted intothe patient. See, e.g. U.S. Pat. Nos. 4,892,538 and 5,283,187. There area variety of techniques available for introducing nucleic acids intoviable cells. The techniques vary depending upon whether the nucleicacid is transferred into cultured cells in vitro, or in vivo in thecells of the intended host. Techniques suitable for the transfer ofnucleic acid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, DEAE-dextran, and calciumphosphate precipitation. A commonly used vector for ex vivo delivery ofa nucleic acid is a retrovirus.

Other in vivo nucleic acid transfer techniques include transfection withviral vectors (such as adenovirus, Herpes simplex I virus, oradeno-associated virus) and lipid-based systems. The nucleic acid andtransfection agent are optionally associated with a microparticle.Exemplary transfection agents include calcium phosphate or calciumchloride co-precipitation, DEAE-dextran-mediated transfection,quaternary ammonium amphiphile DOTMA ((dioleoyloxypropyl)trimethylammonium bromide, commercialized as Lipofectin by GIBCO-BRL))(Felgner et al, (1987) Proc. Natl. Acad. Sci. USA 84, 7413-7417; Maloneet al. (1989) Proc. Natl Acad. Sci. USA 86 6077-6081); lipophilicglutamate diesters with pendent trimethylammonium heads (Ito et al.(1990) Biochem. Biophys. Acta 1023, 124-132); the metabolizable parentlipids such as the cationic lipid dioctadecylamido glycylspermine (DOGS,Transfectam, Promega) and dipalmitoylphosphatidyl ethanolamylspermine(DPPES) (J. P. Behr (1986) Tetrahedron Lett. 27, 5861-5864; J. P. Behret al. (1989) Proc. Natl. Acad. Sci. USA 86, 6982-6986); metabolizablequaternary ammonium salts (DOTB,N-(1-[2,3-dioleoyloxy]propyl)-N,N,N-trimethylammonium methylsulfate(DOTAP) (Boehringer Mannheim), polyethyleneimine (PEI), dioleoyl esters,ChoTB, ChoSC, DOSC) (Leventis et al. (1990) Biochim. Inter. 22,235-241); 3beta[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol(DC-Chol), dioleoylphosphatidyl ethanolamine(DOPE)/3beta[N—(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol DC-Cholin one to one mixtures (Gao et al., (1991) Biochim. Biophys. Acta 1065,8-14), spermine, spermidine, lipopolyamines (Behr et al., BioconjugateChem, 1994, 5: 382-389), lipophilic polylysines (LPLL) (Zhou et al.,(1991) Biochim. Biophys. Acta 939, 8-18),[[(1,1,3,3-tetramethylbutyl)cre-soxy]ethoxy]ethyl]dimethylbenzylammoniumhydroxide (DEBDA hydroxide) with excess phosphatidylcholine/cholesterol(Ballas et al., (1988) Biochim. Biophys. Acta 939, 8-18),cetyltrimethylammonium bromide (CTAB)/DOPE mixtures (Pinnaduwage et al,(1989) Biochim. Biophys. Acta 985, 33-37), lipophilic diester ofglutamic acid (TMAG) with DOPE, CTAB, DEBDA, didodecylammonium bromide(DDAB), and stearylamine in admixture with phosphatidylethanolamine(Rose et al., (1991) Biotechnique 10, 520-525), DDAB/DOPE (TransfectACE,GIBCO BRL), and oligogalactose bearing lipids. Exemplary transfectionenhancer agents that increase the efficiency of transfer include, forexample, DEAE-dextran, polybrene, lysosome-disruptive peptide (Ohmori NI et al, Biochem Biophys Res Commun Jun. 27, 1997; 235(3):726-9),chondroitan-based proteoglycans, sulfated proteoglycans,polyethylenimine, polylysine (Pollard H et al. J Biol Chem, 1998 273(13):7507-11), integrin-binding peptide CYGGRGDTP (SEQ ID NO: 102),linear dextran nonasaccharide, glycerol, cholesteryl groups tethered atthe 3′-terminal internucleoside link of an oligonucleotide (Letsinger,R. L. 1989 Proc Natl Acad Sci USA 86: (17):6553-6), lysophosphatide,lysophosphatidylcholine, lysophosphatidylethanolamine, and 1-oleoyllysophosphatidylcholine.

In some situations it may be desirable to deliver the nucleic acid withan agent that directs the nucleic acid-containing vector to targetcells. Such “targeting” molecules include antibodies specific for acell-surface membrane protein on the target cell, or a ligand for areceptor on the target cell. Where liposomes are employed, proteinswhich bind to a cell-surface membrane protein associated withendocytosis may be used for targeting and/or to facilitate uptake.Examples of such proteins include capsid proteins and fragments thereoftropic for a particular cell type, antibodies for proteins which undergointernalization in cycling, and proteins that target intracellularlocalization and enhance intracellular half-life. In other embodiments,receptor-mediated endocytosis can be used. Such methods are described,for example, in Wu et al., 1987 or Wagner et al., 1990. For review ofthe currently known gene marking and gene therapy protocols, seeAnderson 1992. See also WO 93/25673 and the references cited therein.For additional reviews of gene therapy technology, see Friedmann,Science, 244: 1275-1281 (1989); Anderson, Nature, supplement to vol.392, no 6679, pp. 25-30 (1998); Verma, Scientific American: 68-84(1990); and Miller, Nature, 357: 455-460 (1992).

Screening Methods

Another aspect of the present invention is directed to methods ofidentifying antibodies which modulate (i.e., decrease) activity of aPRLR comprising contacting a PRLR with an antibody, and determiningwhether the antibody modifies activity of the PRLR. The activity in thepresence of the test antibody is compared to the activity in the absenceof the test antibody. Where the activity of the sample containing thetest antibody is lower than the activity in the sample lacking the testantibody, the antibody will have inhibited activity. Effectivetherapeutics depend on identifying efficacious agents devoid ofsignificant toxicity. Antibodies may be screened for binding affinity bymethods known in the art. For example, gel-shift assays, Western blots,radiolabeled competition assay, co-fractionation by chromatography,co-precipitation, cross linking, ELISA, surface plasmon resonance (e.g.,Biacore®), time-resolved fluorometry (e.g., DELFIA) and the like may beused, which are described in, for example, Current Protocols inMolecular Biology (1999), Current Protocols in Immunology (2007) JohnWiley & Sons, NY, which are incorporated herein by reference in theirentirety. In addition, surface plasmon resonance (e.g., Biacore®) may beemployed to assess competition between two antibodies (See, e.g.,Example 7 below). Time-resolved fluorometry (e.g., DELFIA) also may beemployed to assess the level of competition between two antibodies. Forexample, a microplate based competitive screening DELFIA® assay (PerkinElmer) may be performed according to protocols provided by themanufacturer.

To initially screen for antibodies which bind to the desired epitope onthe target antigen, a routine cross-blocking assay such as thatdescribed in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed. Routinecompetitive binding assays may also be used, in which the unknownantibody is characterized by its ability to inhibit binding of target toa target-specific antibody of the invention. Intact antigen, fragmentsthereof such as the extracellular domain, or linear epitopes can beused. Epitope mapping is described in Champe et al., J. Biol. Chem. 270:1388-1394 (1995).

In one variation of an in vitro binding assay, the invention provides amethod comprising the steps of (a) contacting an immobilized PRLR with acandidate antibody and (b) detecting binding of the candidate antibodyto the PRLR. In an alternative embodiment, the candidate antibody isimmobilized and binding of PRLR is detected. Immobilization isaccomplished using any of the methods well known in the art, includingcovalent bonding to a support, a bead, or a chromatographic resin, aswell as non-covalent, high affinity interaction such as antibodybinding, or use of streptavidin/biotin binding wherein the immobilizedcompound includes a biotin moiety. Detection of binding can beaccomplished (i) using a radioactive label on the compound that is notimmobilized, (ii) using a fluorescent label on the non-immobilizedcompound, (iii) using an antibody immunospecific for the non-immobilizedcompound, (iv) using a label on the non-immobilized compound thatexcites a fluorescent support to which the immobilized compound isattached, as well as other techniques well known and routinely practicedin the art.

Antibodies that modulate (i.e., increase, decrease, or block) theactivity of the target antigen may be identified by incubating acandidate antibody with target antigen (or a cell expressing targetantigen) and determining the effect of the candidate antibody on theactivity or expression of the target antigen. The activity in thepresence of the test antibody is compared to the activity in the absenceof the test antibody. Where the activity of the sample containing thetest antibody is lower than the activity in the sample lacking the testantibody, the antibody will have inhibited activity. The selectivity ofan antibody that modulates the activity of a target antigen polypeptideor polynucleotide can be evaluated by comparing its effects on thetarget antigen to its effect on other related compounds.

In particular exemplary embodiments, it is contemplated that theantibodies are tested for their effect in a cultured cell system todetermine their ability to prevent PRLR dimerization and/or neutralizePRLR in inducing STAT5 and/or MAPK and/or AKT phosphorylation or otherindicators of PRLR signaling. Additionally, cellular assays includingproliferation assays, soft agar assays, and/or cytotoxicity assays asdescribed herein may be used to evaluate a particular PRLR antibody.

The biological activity of a particular antibody, or combination ofantibodies, may be evaluated in vivo using a suitable animal model. Forexample, xenogenic cancer models wherein human cancer cells areintroduced into immune compromised animals, such as nude or SCID mice,may be used. Efficacy may be predicted using assays which measureinhibition of tumor formation, tumor regression or metastasis, and thelike.

The invention also comprehends high throughput screening (HTS) assays toidentify antibodies that interact with or inhibit biological activity(i.e., inhibit enzymatic activity, binding activity, intracellularsignaling, etc.) of target antigen. HTS assays permit screening of largenumbers of compounds in an efficient manner. Cell-based HTS systems arecontemplated to investigate the interaction between target antigen andits binding partners. HTS assays are designed to identify “hits” or“lead compounds” having the desired property, from which modificationscan be designed to improve the desired property.

In another embodiment of the invention, high throughput screening forantibody fragments or CDRs with 1, 2, 3 or more modifications to aminoacids within the CDRs having suitable binding affinity to a targetantigen polypeptide is employed.

Combination Therapy

Having identified more than one antibody that is effective in an animalmodel, it may be further advantageous to mix two or more such antibodiestogether (which bind to the same or different target antigens) toprovide still improved efficacy. Compositions comprising one or moreantibody may be administered to persons or mammals suffering from, orpredisposed to suffer from, cancer. Concurrent administration of twotherapeutic agents does not require that the agents be administered atthe same time or by the same route, as long as there is an overlap inthe time period during which the agents are exerting their therapeuticeffect. Simultaneous or sequential administration is contemplated, as isadministration on different days or weeks.

Although antibody therapy may be useful for all stages of cancers,antibody therapy may be particularly appropriate in advanced ormetastatic cancers. Combining the antibody therapy method with achemotherapeutic or radiation regimen may be preferred in patients thathave not received chemotherapeutic treatment, whereas treatment with theantibody therapy may be indicated for patients who have received one ormore chemotherapies. Additionally, antibody therapy can also enable theuse of reduced dosages of concomitant chemotherapy, particularly inpatients that do not tolerate the toxicity of the chemotherapeutic agentvery well.

The methods of the invention contemplate the administration of singleantibodies, as well as combinations, or “cocktails”, of differentantibodies. Such antibody cocktails may have certain advantages inasmuchas they contain antibodies which exploit different effector mechanismsor combine directly cytotoxic antibodies with antibodies that rely onimmune effector functionality. Such antibodies in combination mayexhibit synergistic therapeutic effects. By way of example, the methodsof the invention contemplate administering antibodies to M-CSF, RANKL,Taxotere™, Herceptin™, Avastin™, Erbitux™ or anti-EGFR antibodies, andTamoxifen.

A cytotoxic agent refers to a substance that inhibits or prevents thefunction of cells and/or causes destruction of cells. The term isintended to include radioactive isotopes (e.g., I¹³¹, I¹²⁵, Y⁹⁰ andRe¹⁸⁶), chemotherapeutic agents, and toxins such as enzymatically activetoxins of bacterial, fungal, plant or animal origin or synthetic toxins,or fragments thereof. A non-cytotoxic agent refers to a substance thatdoes not inhibit or prevent the function of cells and/or does not causedestruction of cells. A non-cytotoxic agent may include an agent thatcan be activated to be cytotoxic. A non-cytotoxic agent may include abead, liposome, matrix or particle (see, e.g., U.S. Patent Publications2003/0028071 and 2003/0032995 which are incorporated by referenceherein). Such agents may be conjugated, coupled, linked or associatedwith an antibody according to the invention.

Cancer chemotherapeutic agents include, without limitation, alkylatingagents, such as carboplatin and cisplatin; nitrogen mustard alkylatingagents; nitrosourea alkylating agents, such as carmustine (BCNU);antimetabolites, such as methotrexate; folinic acid; purine analogantimetabolites, mercaptopurine; pyrimidine analog antimetabolites, suchas fluorouracil (5-FU) and gemcitabine (Gemzar®); hormonalantineoplastics, such as goserelin, leuprolide, and tamoxifen; naturalantineoplastics, such as aldesleukin, interleukin-2, docetaxel,etoposide (VP-16), interferon alfa, paclitaxel (Taxol®), and tretinoin(ATRA); antibiotic natural antineoplastics, such as bleomycin,dactinomycin, daunorubicin, doxorubicin, daunomycin and mitomycinsincluding mitomycin C; and vinca alkaloid natural antineoplastics, suchas vinblastine, vincristine, vindesine; hydroxyurea; aceglatone,adriamycin, ifosfamide, enocitabine, epitiostanol, aclarubicin,ancitabine, nimustine, procarbazine hydrochloride, carboquone,carboplatin, carmofur, chromomycin A3, antitumor polysaccharides,antitumor platelet factors, cyclophosphamide (Cytoxin®), Schizophyllan,cytarabine (cytosine arabinoside), dacarbazine, thioinosine, thiotepa,tegafur, dolastatins, dolastatin analogs such as auristatin, CPT-11(irinotecan), mitozantrone, vinorelbine, teniposide, aminopterin,caminomycin, esperamicins (See, e.g., U.S. Pat. No. 4,675,187),neocarzinostatin, OK-432, bleomycin, furtulon, broxuridine, busulfan,honvan, peplomycin, bestatin (Ubenimex®), interferon-β, mepitiostane,mitobronitol, melphalan, laminin peptides, lentinan, Coriolus versicolorextract, tegafur/uracil, estramustine (estrogen/mechlorethamine).

Further, additional agents used as therapy for cancer patients includeEPO, G-CSF, ganciclovir; antibiotics, leuprolide; meperidine; zidovudine(AZT); interleukins 1 through 18, including mutants and analogues;interferons or cytokines, such as interferons α, β, and γ hormones, suchas luteinizing hormone releasing hormone (LHRH) and analogues and,gonadotropin releasing hormone (GnRH); growth factors, such astransforming growth factor-β (TGF-β), fibroblast growth factor (FGF),nerve growth factor (NGF), growth hormone releasing factor (GHRF),epidermal growth factor (EGF), fibroblast growth factor homologousfactor (FGFHF), hepatocyte growth factor (HGF), and insulin growthfactor (IGF); tumor necrosis factor-α & β (TNF-α & β); invasioninhibiting factor-2 (IIF-2); bone morphogenetic proteins 1-7 (BMP 1-7);somatostatin; thymosin-α-1; γ-globulin; superoxide dismutase (SOD); EGFR(epidermal growth factor receptor) antagonists, such as for exampleCetuximab and Gefitinib; PR (progesterone receptor) antagonists andmodulators such as Mifepristone and Onapristone™; aromatoase inhibitorssuch as for example Anastrozole, Exemestane and Letrozole; anti-estrogenagents, estrogen receptor anatagonists and modulators, such as forexample Tamoxifen, Toremifene and Fulvestrant; complement factors;anti-angiogenesis factors; antigenic materials; and pro-drugs.

Prodrug refers to a precursor or derivative form of a pharmaceuticallyactive substance that is less cytotoxic or non-cytotoxic to tumor cellscompared to the parent drug and is capable of being enzymaticallyactivated or converted into an active or the more active parent form.See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” Biochemical SocietyTransactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stellaet al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery,”Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, HumanaPress (1985). Prodrugs include, but are not limited to,phosphate-containing prodrugs, thiophosphate-containing prodrugs,sulfate-containing prodrugs, peptide-containing prodrugs, D-aminoacid-modified prodrugs, glycosylated prodrugs, β-lactam-containingprodrugs, optionally substituted phenoxyacetamide-containing prodrugs oroptionally substituted phenylacetamide-containing prodrugs,5-fluorocytosine and other 5-fluorouridine prodrugs which can beconverted into the more active cytotoxic free drug. Examples ofcytotoxic drugs that can be derivatized into a prodrug form for useherein include, but are not limited to, those chemotherapeutic agentsdescribed above.

Administration and Preparation

The antibodies of the invention may be formulated into pharmaceuticalcompositions comprising a carrier suitable for the desired deliverymethod. Suitable carriers include any material which, when combined withantibodies, retains the desired activity of the antibody and isnonreactive with the subject's immune systems. Examples include, but arenot limited to, any of a number of standard pharmaceutical carriers suchas sterile phosphate buffered saline solutions, bacteriostatic water,and the like. A variety of aqueous carriers may be used, e.g., water,buffered water, 0.4% saline, 0.3% glycine and the like, and may includeother proteins for enhanced stability, such as albumin, lipoprotein,globulin, etc., subjected to mild chemical modifications or the like.

Therapeutic formulations of the antibody are prepared for storage bymixing the antibody having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

The antibody is administered by any suitable means, includingparenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local treatment, intralesionaladministration. Parenteral infusions include intravenous, intraarterial,intraperitoneal, intramuscular, intradermal or subcutaneousadministration. In addition, the antibody is suitably administered bypulse infusion, particularly with declining doses of the antibody.Preferably the dosing is given by injections, most preferablyintravenous or subcutaneous injections. Other administration methods arecontemplated, including topical, particularly transdermal, transmucosal,rectal, oral or local administration e.g. through a catheter placedclose to the desired site.

For nasal administration, the pharmaceutical formulations andmedicaments may be a spray or aerosol containing an appropriatesolvent(s) and optionally other compounds such as, but not limited to,stabilizers, antimicrobial agents, antioxidants, pH modifiers,surfactants, bioavailability modifiers and combinations of these. Apropellant for an aerosol formulation may include compressed air,nitrogen, carbon dioxide, or a hydrocarbon based low boiling solvent.

Injectable dosage forms generally include aqueous suspensions or oilsuspensions which may be prepared using a suitable dispersant or wettingagent and a suspending agent. Injectable forms may be in solution phaseor in the form of a suspension, which is prepared with a solvent ordiluent. Acceptable solvents or vehicles include sterilized water,Ringer's solution, or an isotonic aqueous saline solution.

For injection, the pharmaceutical formulation and/or medicament may be apowder suitable for reconstitution with an appropriate solution asdescribed above. Examples of these include, but are not limited to,freeze dried, rotary dried or spray dried powders, amorphous powders,granules, precipitates, or particulates. For injection, the formulationsmay optionally contain stabilizers, pH modifiers, surfactants,bioavailability modifiers and combinations of these.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsule. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and yethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the Lupron Depot™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods. When encapsulated antibodies remainin the body for a long time, they may denature or aggregate as a resultof exposure to moisture at 37° C., resulting in a loss of biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be intermolecularS—S bond formation through thio-disulfide interchange, stabilization maybe achieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions. Other strategiesknown in the art may be used.

The formulations of the invention may be designed to be short-acting,fast-releasing, long-acting, or sustained-releasing as described herein.Thus, the pharmaceutical formulations may also be formulated forcontrolled release or for slow release.

The instant compositions may also comprise, for example, micelles orliposomes, or some other encapsulated form, or may be administered in anextended release form to provide a prolonged storage and/or deliveryeffect. Therefore, the pharmaceutical formulations and medicaments maybe compressed into pellets or cylinders and implanted intramuscularly orsubcutaneously as depot injections or as implants such as stents. Suchimplants may employ known inert materials such as silicones andbiodegradable polymers.

Besides those representative dosage forms described above,pharmaceutically acceptable excipients and carries are generally knownto those skilled in the art and are thus included in the instantinvention. Such excipients and carriers are described, for example, in“Remingtons Pharmaceutical Sciences” Mack Pub. Co., New Jersey (1991),which is incorporated herein by reference.

Specific dosages may be adjusted depending on conditions of disease, theage, body weight, general health conditions, genotype, sex, and diet ofthe subject, dose intervals, administration routes, excretion rate, andcombinations of drugs. Any of the above dosage forms containingeffective amounts are well within the bounds of routine experimentationand therefore, well within the scope of the instant invention.

Antibodies of the invention will often be prepared substantially free ofother naturally occurring immunoglobulins or other biological molecules.Preferred antibodies will also exhibit minimal toxicity whenadministered to a mammal afflicted with, or predisposed to suffer fromcancer.

The compositions of the invention may be sterilized by conventional,well known sterilization techniques. The resulting solutions may bepackaged for use or filtered under aseptic conditions and lyophilized,the lyophilized preparation being combined with a sterile solution priorto administration. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents and the like, for example, sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride andstabilizers (e.g., 120% maltose, etc.).

The antibodies of the present invention may also be administered vialiposomes, which are small vesicles composed of various types of lipidsand/or phospholipids and/or surfactant which are useful for delivery ofa drug (such as the antibodies disclosed herein and, optionally, achemotherapeutic agent). Liposomes include emulsions, foams, micelles,insoluble monolayers, phospholipid dispersions, lamellar layers and thelike, and can serve as vehicles to target the antibodies to a particulartissue as well as to increase the half life of the composition. Avariety of methods are available for preparing liposomes, as describedin, e.g., U.S. Pat. Nos. 4,837,028 and 5,019,369, which patents areincorporated herein by reference.

Liposomes containing the antibody are prepared by methods known in theart, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030 (1980);and U.S. Pat. Nos. 4,485,045 and 4,544,545.

Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556. Particularly useful liposomes can be generated by the reversephase evaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem. 257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome [see, e.g., Gabizon et al., J. National Cancer Inst.81(19): 1484 (1989)].

The concentration of antibody in these compositions can vary widely,i.e., from less than about 10%, usually at least about 25% to as much as75% or 90% by weight and will be selected primarily by fluid volumes,viscosities, etc., in accordance with the particular mode ofadministration selected. Actual methods for preparing orally, topicallyand parenterally administrable compositions will be known or apparent tothose skilled in the art and are described in detail in, for example,Remington's Pharmaceutical Science, 19th ed., Mack Publishing Co.,Easton, Pa. (1995), which is incorporated herein by reference.

Determination of an effective amount of a composition of the inventionto treat disease in a patient can be accomplished through standardempirical methods which are well known in the art.

Compositions of the invention are administered to a mammal alreadysuffering from, or predisposed to or at risk of, for example, breast,prostate, or lung cancer, in an amount sufficient to prevent or at leastpartially arrest the development of disease. An amount adequate toaccomplish this is defined as a “therapeutically effective dose.”Effective amounts of an antibody will vary and depend on the severity ofthe disease and the weight and general state of the patient beingtreated, but generally range from about 1.0 μg/kg to about 100 mg/kgbody weight. Exemplary doses may range from about 10 μg/kg to about 30mg/kg, or from about 0.1 mg/kg to about 20 mg/kg or from about 1 mg/kgto about 10 mg/kg per application. Antibody may also be dosed by bodysurface area (e.g. up to 4.5 g/square meter). Other exemplary doses ofantibody include up to 8 g total in a single administration (assuming abody weight of 80 kg or body surface area of 1.8 square meters).

Administration may be by any means known in the art. For example,antibody may be administered by one or more separate bolusadministrations, or by short or long term infusion over a period of,e.g., 5, 10, 15, 30, 60, 90, 120 minutes or more. Following an initialtreatment period, and depending on the patient's response and toleranceof the therapy, maintenance doses may be administered, e.g., weekly,biweekly, every 3 weeks, every 4 weeks, monthly, bimonthly, every 3months, or every 6 months, as needed to maintain patient response. Morefrequent dosages may be needed until a desired suppression of diseasesymptoms occurs, and dosages may be adjusted as necessary. The progressof this therapy is easily monitored by conventional techniques andassays. The therapy may be for a defined period or may be chronic andcontinue over a period of years until disease progression or death.

Single or multiple administrations of the compositions can be carriedout with the dose levels and pattern being selected by the treatingphysician. For the prevention or treatment of disease, the appropriatedosage of antibody will depend on the type of disease to be treated, asdefined above, the severity and course of the disease, whether theantibody is administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to theantibody, and the discretion of the attending physician. The antibody issuitably administered to the patient at one time or over a series oftreatments.

In any event, the formulations should provide a quantity of therapeuticantibody over time that is sufficient to exert the desired biologicalactivity, e.g. prevent or minimize the severity of cancer. Thecompositions of the present invention may be administered alone or as anadjunct therapy in conjunction with other therapeutics known in the artfor the treatment of such diseases.

The antibody composition will be formulated, dosed, and administered ina fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Thetherapeutically effective amount of the antibody to be administered willbe governed by such considerations, and is the minimum amount necessaryto prevent, ameliorate, or treat the target-mediated disease, conditionor disorder. Such amount is preferably below the amount that is toxic tothe host or renders the host significantly more susceptible toinfections.

The antibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disease, condition or disorderor treatment, and other factors discussed above. These are generallyused in the same dosages and with administration routes as usedhereinbefore or about from 1 to 99% of the heretofore employed dosages.

In another embodiment of the invention, there is provided an article ofmanufacture containing materials useful for the treatment of the desiredcondition. The article of manufacture comprises a container and a label.Suitable containers include, for example, bottles, vials, syringes, andtest tubes. The containers may be formed from a variety of materialssuch as glass or plastic. The container holds a composition which iseffective for treating the condition and may have a sterile access port(for example the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). Theactive agent in the composition is the antibody of the invention. Thelabel on, or associated with, the container indicates that thecomposition is used for treating the condition of choice. The article ofmanufacture may further comprise a second container containing a secondtherapeutic agent (including any of the second therapeutic agents fordiseases discussed herein or known in the art). The article ofmanufacture may further comprise another container containing apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution or dextrose solution for reconstituting a lyophilizedantibody formulation. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, syringes, and package inserts withinstructions for use.

Immunotherapy

Antibodies useful in treating patients having cancers include thosewhich are capable of initiating a potent immune response against thetumor and those which are capable of direct cytotoxicity. Antibodiesconjugated to cytotoxic agents may be used to target the cytotoxicagents to tumor tissues expressing PRLR. Alternatively, antibodies mayelicit tumor cell lysis by either complement-mediated orantibody-dependent cell cytotoxicity (ADCC) mechanisms, both of whichrequire an intact Fc portion of the immunoglobulin molecule forinteraction with effector cell Fc receptor sites or complement proteins.In addition, antibodies that exert a direct biological effect on tumorgrowth are useful in the practice of the invention. Potential mechanismsby which such directly cytotoxic antibodies may act include inhibitionof cell growth, modulation of cellular differentiation, modulation oftumor angiogenesis factor profiles, and the induction of apoptosis. Themechanism by which a particular antibody exerts an anti-tumor effect maybe evaluated using any number of in vitro assays designed to determineADCC, ADMMC, complement-mediated cell lysis, and so forth, as isgenerally known in the art.

In one embodiment, immunotherapy is carried out using antibodies thatbind to PRLR and inhibit activation of PRLR.

Anti-PRLR antibodies may be administered in their “naked” orunconjugated form, or may be conjugated directly to other therapeutic ordiagnostic agents, or may be conjugated indirectly to carrier polymerscomprising such other therapeutic or diagnostic agents.

Antibodies can be detectably labeled through the use of radioisotopes,affinity labels (such as biotin, avidin, etc.), enzymatic labels (suchas horseradish peroxidase, alkaline phosphatase, etc.) fluorescent orluminescent or bioluminescent labels (such as FITC or rhodamine, etc.),paramagnetic atoms, and the like. Procedures for accomplishing suchlabeling are well known in the art; for example, see (Sternberger, L. A.et al., J. Histochem. Cytochem. 18:315 (1970); Bayer, E. A. et al.,Meth. Enzym. 62:308 (1979); Engval, E. et al., Immunol. 109:129 (1972);Goding, J. W. J. Immunol. Meth. 13:215 (1976)).

Conjugation of antibody moieties is described in U.S. Pat. No.6,306,393. General techniques are also described in Shih et al., Int. J.Cancer 41:832-839 (1988); Shih et al., Int. J. Cancer 46:1101-1106(1990); and Shih et al., U.S. Pat. No. 5,057,313. This general methodinvolves reacting an antibody component having an oxidized carbohydrateportion with a carrier polymer that has at least one free amine functionand that is loaded with a plurality of drug, toxin, chelator, boronaddends, or other therapeutic agent. This reaction results in an initialSchiff base (imine) linkage, which can be stabilized by reduction to asecondary amine to form the final conjugate.

The carrier polymer may be, for example, an aminodextran or polypeptideof at least 50 amino acid residues. Various techniques for conjugating adrug or other agent to the carrier polymer are known in the art. Apolypeptide carrier can be used instead of aminodextran, but thepolypeptide carrier should have at least 50 amino acid residues in thechain, preferably 100-5000 amino acid residues. At least some of theamino acids should be lysine residues or glutamate or aspartateresidues. The pendant amines of lysine residues and pendant carboxylatesof glutamine and aspartate are convenient for attaching a drug, toxin,immunomodulator, chelator, boron addend or other therapeutic agent.Examples of suitable polypeptide carriers include polylysine,polyglutamic acid, polyaspartic acid, co-polymers thereof, and mixedpolymers of these amino acids and others, e.g., serines, to conferdesirable solubility properties on the resultant loaded carrier andconjugate.

Alternatively, conjugated antibodies can be prepared by directlyconjugating an antibody component with a therapeutic agent. The generalprocedure is analogous to the indirect method of conjugation except thata therapeutic agent is directly attached to an oxidized antibodycomponent. For example, a carbohydrate moiety of an antibody can beattached to polyethyleneglycol to extend half-life.

Alternatively, a therapeutic agent can be attached at the hinge regionof a reduced antibody component via disulfide bond formation, or using aheterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J. Cancer 56:244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, Chemistry Of Protein Conjugation andCross-Linking (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in Monoclonal Antibodies: Principlesand Applications, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in Monoclonal Antibodies: Production,Enineering and Clinical Application, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). A variety of bifunctional proteincoupling agents are known in the art, such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene).

Finally, fusion proteins can be constructed that comprise one or moreanti-PRLR antibody moieties and another polypeptide. Methods of makingantibody fusion proteins are well known in the art. See, e.g., U.S. Pat.No. 6,306,393. Antibody fusion proteins comprising an interleukin-2moiety are described by Boleti et al., Ann. Oncol. 6:945 (1995), Nicoletet al., Cancer Gene Ther. 2:161 (1995), Becker et al., Proc. Nat'l Acad.Sci. USA 93:7826 (1996), Hank et al., Clin. Cancer Res. 2:1951 (1996),and Hu et al., Cancer Res. 56:4998 (1996).

In one embodiment, the antibodies of the invention are used as aradiosensitizer. In such embodiments, the antibodies are conjugated to aradiosensitizing agent. The term “radiosensitizer,” as used herein, isdefined as a molecule, preferably a low molecular weight molecule,administered to animals in therapeutically effective amounts to increasethe sensitivity of the cells to be radiosensitized to electromagneticradiation and/or to promote the treatment of diseases that are treatablewith electromagnetic radiation. Diseases that are treatable withelectromagnetic radiation include neoplastic diseases, benign andmalignant tumors, and cancerous cells.

The terms “electromagnetic radiation” and “radiation” as used hereininclude, but are not limited to, radiation having the wavelength of10⁻²⁰ to 100 meters. Preferred embodiments of the present inventionemploy the electromagnetic radiation of: gamma-radiation (10⁻²⁰ to 10⁻¹³m), X-ray radiation (10⁻¹² to 10⁻⁹ m), ultraviolet light (10 nm to 400nm), visible light (400 nm to 700 nm), infrared radiation (700 nm to 1.0mm), and microwave radiation (1 mm to 30 cm).

Radiosensitizers are known to increase the sensitivity of cancerouscells to the toxic effects of electromagnetic radiation. Many cancertreatment protocols currently employ radiosensitizers activated by theelectromagnetic radiation of X-rays. Examples of X-ray activatedradiosensitizers include, but are not limited to, the following:metronidazole, misonidazole, desmethylmisonidazole, pimonidazole,etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, E09, RB 6145,nicotinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR),bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea, cisplatin,and therapeutically effective analogs and derivatives of the same.

Photodynamic therapy (PDT) of cancers employs visible light as theradiation activator of the sensitizing agent. Examples of photodynamicradiosensitizers include the following, but are not limited to:hematoporphyrin derivatives, Photofrin(r), benzoporphyrin derivatives,NPe6, tin etioporphyrin (SnET2), pheoborbide-a, bacteriochlorophyll-a,naphthalocyanines, phthalocyanines, zinc phthalocyanine, andtherapeutically effective analogs and derivatives of the same.

In another embodiment, the antibody may be conjugated to a receptor(such streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the patient, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a ligand (e.g., avidin) which is conjugatedto a cytotoxic agent (e.g., a radionuclide).

“Label” refers to a detectable compound or composition which isconjugated directly or indirectly to the antibody. The label may itselfbe detectable by itself (e.g., radioisotope labels or fluorescentlabels) or, in the case of an enzymatic label, may catalyze chemicalalteration of a substrate compound or composition which is detectable.Alternatively, the label may not be detectable on its own but may be anelement that is bound by another agent that is detectable (e.g. anepitope tag or one of a binding partner pair such as biotin-avidin,etc.) Thus, the antibody may comprise a label or tag that facilitatesits isolation, and methods of the invention to identify antibodiesinclude a step of isolating the antibody through interaction with thelabel or tag.

Exemplary therapeutic immunoconjugates comprise the antibody describedherein conjugated to a cytotoxic agent such as a chemotherapeutic agent,toxin (e.g., an enzymatically active toxin of bacterial, fungal, plantor animal origin, or fragments thereof), or a radioactive isotope (i.e.,a radioconjugate). Fusion proteins are described in further detailbelow.

Chelators for radiometals or magnetic resonance enhancers are well-knownin the art. Typical are derivatives of ethylenediaminetetraacetic acid(EDTA) and diethylenetriaminepentaacetic acid (DTPA). These chelatorstypically have groups on the side chain by which the chelator can beattached to a carrier. Such groups include, e.g., benzylisothiocyanate,by which the DTPA or EDTA can be coupled to the amine group of acarrier. Alternatively, carboxyl groups or amine groups on a chelatorcan be coupled to a carrier by activation or prior derivatization andthen coupling, all by well-known means.

Boron addends, such as carboranes, can be attached to antibodycomponents by conventional methods. For example, carboranes can beprepared with carboxyl functions on pendant side chains, as is wellknown in the art. Attachment of such carboranes to a carrier, e.g.,aminodextran, can be achieved by activation of the carboxyl groups ofthe carboranes and condensation with amines on the carrier to produce anintermediate conjugate. Such intermediate conjugates are then attachedto antibody components to produce therapeutically usefulimmunoconjugates, as described below.

A polypeptide carrier can be used instead of aminodextran, but thepolypeptide carrier should have at least 50 amino acid residues in thechain, preferably 100-5000 amino acid residues. At least some of theamino acids should be lysine residues or glutamate or aspartateresidues. The pendant amines of lysine residues and pendant carboxylatesof glutamine and aspartate are convenient for attaching a drug, toxin,immunomodulator, chelator, boron addend or other therapeutic agent.Examples of suitable polypeptide carriers include polylysine,polyglutamic acid, polyaspartic acid, co-polymers thereof, and mixedpolymers of these amino acids and others, e.g., serines, to conferdesirable solubility properties on the resultant loaded carrier andimmunoconjugate.

Conjugation of the intermediate conjugate with the antibody component iseffected by oxidizing the carbohydrate portion of the antibody componentand reacting the resulting aldehyde (and ketone) carbonyls with aminegroups remaining on the carrier after loading with a drug, toxin,chelator, immunomodulator, boron addend, or other therapeutic agent.Alternatively, an intermediate conjugate can be attached to an oxidizedantibody component via amine groups that have been introduced in theintermediate conjugate after loading with the therapeutic agent.Oxidation is conveniently effected either chemically, e.g., with NaIO₄or other glycolytic reagent, or enzymatically, e.g., with neuraminidaseand galactose oxidase. In the case of an aminodextran carrier, not allof the amines of the aminodextran are typically used for loading atherapeutic agent. The remaining amines of aminodextran condense withthe oxidized antibody component to form Schiff base adducts, which arethen reductively stabilized, normally with a borohydride reducing agent.

Analogous procedures are used to produce other immunoconjugatesaccording to the invention. Loaded polypeptide carriers preferably havefree lysine residues remaining for condensation with the oxidizedcarbohydrate portion of an antibody component. Carboxyls on thepolypeptide carrier can, if necessary, be converted to amines by, e.g.,activation with DCC and reaction with an excess of a diamme.

The final immunoconjugate is purified using conventional techniques,such as sizing chromatography on Sephacryl S-300 or affinitychromatography using one or more CD84Hy epitopes.

Alternatively, immunoconjugates can be prepared by directly conjugatingan antibody component with a therapeutic agent. The general procedure isanalogous to the indirect method of conjugation except that atherapeutic agent is directly attached to an oxidized antibodycomponent.

It will be appreciated that other therapeutic agents can be substitutedfor the chelators described herein. Those of skill in the art will beable to devise conjugation schemes without undue experimentation.

As a further illustration, a therapeutic agent can be attached at thehinge region of a reduced antibody component via disulfide bondformation. For example, the tetanus toxoid peptides can be constructedwith a single cysteine residue that is used to attach the peptide to anantibody component. As an alternative, such peptides can be attached tothe antibody component using a heterobifunctional cross-linker, such asN-succinyl 3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J.Cancer 56:244 (1994). General techniques for such conjugation arewell-known in the art. See, for example, Wong, Chemistry Of ProteinConjugation and Cross-Linking (CRC Press 1991); Upeslacis et al.,“Modification of Antibodies by Chemical Methods,” in MonoclonalAntibodies: Principles and Applications, Birch et al. (eds.), pages187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterizationof Synthetic Peptide-Derived Antibodies,” in Monoclonal Antibodies:Production, Enineering and Clinical Application, Ritter et al. (eds.),pages 60-84 (Cambridge University Press 1995).

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such asbis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionuclide to the antibody (see, e.g., WO94/11026).

As described above, carbohydrate moieties in the Fc region of anantibody can be used to conjugate a therapeutic agent. However, the Fcregion may be absent if an antibody fragment is used as the antibodycomponent of the immunoconjugate. Nevertheless, it is possible tointroduce a carbohydrate moiety into the light chain variable region ofan antibody or antibody fragment. See, for example, Leung et al., J.Immunol. 154:5919 (1995); Hansen et al., U.S. Pat. No. 5,443,953. Theengineered carbohydrate moiety is then used to attach a therapeuticagent.

In addition, those of skill in the art will recognize numerous possiblevariations of the conjugation methods. For example, the carbohydratemoiety can be used to attach polyethyleneglycol in order to extend thehalf-life of an intact antibody, or antigen-binding fragment thereof, inblood, lymph, or other extracellular fluids. Moreover, it is possible toconstruct a “divalent immunoconjugate” by attaching therapeutic agentsto a carbohydrate moiety and to a free sulfhydryl group. Such a freesulfhydryl group may be located in the hinge region of the antibodycomponent.

Antibody Fusion Proteins

The present invention contemplates the use of fusion proteins comprisingone or more antibody moieties and another polypeptide, such as animmunomodulator or toxin moiety. Methods of making antibody fusionproteins are well known in the art. See, e.g., U.S. Pat. No. 6,306,393.Antibody fusion proteins comprising an interleukin-2 moiety aredescribed by Boleti et al., Ann. Oncol. 6:945 (1995), Nicolet et al.,Cancer Gene Ther. 2:161 (1995), Becker et al., Proc. Nat'l Acad. Sci.USA 93:7826 (1996), Hank et al., Clin. Cancer Res. 2:1951 (1996), and Huet al., Cancer Res. 56:4998 (1996). In addition, Yang et al., Hum.Antibodies Hybridomas 6:129 (1995), describe a fusion protein thatincludes an F(ab′)₂ fragment and a tumor necrosis factor alpha moiety.

Methods of making antibody-toxin fusion proteins in which a recombinantmolecule comprises one or more antibody components and a toxin orchemotherapeutic agent also are known to those of skill in the art. Forexample, antibody-Pseudomonas exotoxin A fusion proteins have beendescribed by Chaudhary et al., Nature 339:394 (1989), Brinkmann et al.,Proc. Nat'l Acad. Sci. USA 88:8616 (1991), Batra et al., Proc. Nat'lAcad. Sci. USA 89:5867 (1992), Friedman et al., J. Immunol. 150:3054(1993), Wels et al., Int. J. Can. 60:137 (1995), Fominaya et al., J.Biol. Chem. 271:10560 (1996), Kuan et al., Biochemistry 35:2872 (1996),and Schmidt et al., Int. J. Can. 65:538 (1996). Antibody-toxin fusionproteins containing a diphtheria toxin moiety have been described byKreitman et al., Leukemia 7:553 (1993), Nicholls et al., J. Biol. Chem.268:5302 (1993), Thompson et al., J. Biol. Chem. 270:28037 (1995), andVallera et al., Blood 88:2342 (1996). Deonarain et al., Tumor Targeting1:177 (1995), have described an antibody-toxin fusion protein having anRNase moiety, while Linardou et al., Cell Biophys. 24-25:243 (1994),produced an antibody-toxin fusion protein comprising a DNase Icomponent. Gelonin was used as the toxin moiety in the antibody-toxinfusion protein of Wang et al., Abstracts of the 209th ACS NationalMeeting, Anaheim, Calif., Apr. 2-6, 1995, Part 1, BIOT005. As a furtherexample, Dohlsten et al., Proc. Nat'l Acad. Sci. USA 91:8945 (1994),reported an antibody-toxin fusion protein comprising Staphylococcalenterotoxin-A.

Illustrative of toxins which are suitably employed in the preparation ofsuch conjugates are ricin, abrin, ribonuclease, DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtherin toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin. See, for example,Pastan et al., Cell 47:641 (1986), and Goldenberg, C A—A Cancer Journalfor Clinicians 44:43 (1994). Other suitable toxins are known to those ofskill in the art.

Antibodies of the present invention may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g., a peptidyl chemotherapeutic agent, See WO81/01145) to anactive anti-cancer drug. See, for example, WO88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to covertit into its more active, cytotoxic form.

Enzymes that are useful in the method of this invention include, but arenot limited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs; β-lactamaseuseful for converting drugs derivatized with β-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as abzymes, can be used to convert the prodrugs of theinvention into free active drugs (See, e.g., Massey, Nature 328: 457-458(1987)). Antibody-abzyme conjugates can be prepared as described hereinfor delivery of the abzyme to a tumor cell population.

The enzymes of this invention can be covalently bound to the antibodiesby techniques well known in the art such as the use of theheterobifunctional crosslinking reagents discussed above. Alternatively,fusion proteins comprising at least the antigen binding region of anantibody of the invention linked to at least a functionally activeportion of an enzyme of the invention can be constructed usingrecombinant DNA techniques well known in the art (See, e.g., Neubergeret al., Nature 312: 604-608 (1984))

Non-Therapeutic Uses

The antibodies of the invention may be used as affinity purificationagents for target antigen or in diagnostic assays for target antigen,e.g., detecting its expression in specific cells, tissues, or serum. Theantibodies may also be used for in vivo diagnostic assays. Generally,for these purposes the antibody is labeled with a radionuclide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, such as ELISAs, and immunoprecipitation assays. Zola,Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press,Inc. 1987). The antibodies may also be used for immunohistochemistry, tolabel tumor samples using methods known in the art.

As a matter of convenience, the antibody of the present invention can beprovided in a kit, i.e., a packaged combination of reagents inpredetermined amounts with instructions for performing the diagnosticassay. Where the antibody is labeled with an enzyme, the kit willinclude substrates and cofactors required by the enzyme (e.g., asubstrate precursor which provides the detectable chromophore orfluorophore). In addition, other additives may be included such asstabilizers, buffers (e.g., a block buffer or lysis buffer) and thelike. The relative amounts of the various reagents may be varied widelyto provide for concentrations in solution of the reagents whichsubstantially optimize the sensitivity of the assay. Particularly, thereagents may be provided as dry powders, usually lyophilized, includingexcipients which on dissolution will provide a reagent solution havingthe appropriate concentration.

The invention is illustrated by the following examples, which are notintended to be limiting in any way.

EXAMPLES Example 1 Preparation of ECD, S1 and S2 Domain Fragments ofPRLR

Recombinant expression and purification of fragments of PRLRcorresponding to the extracellular domain (ECD, amino acids 25-234 ofSEQ ID NO: 2), the S1 domain (amino acids 25-125 of SEQ ID NO: 2), andthe S2 domain (amino acids 126-234 of SEQ ID NO: 2) of PRLR was carriedout as follows. Expression constructs for insect expression of ECD, S1and S2 were designed as shown in Table 2, and primers were designed forcloning the fragments based on their respective amino acid sequences (asshown in Table 3).

TABLE 2

TABLE 3 PCR primers used (F = forward; Domain R = Reverse) PCR primersequences ECD F1 SEQ ID NO: 3 GGGACAAGTTTGTACAAAAA AGCAGGCTACGAAGGAGATATACATATGAAGGAAAATGTG GCATCTGCAA R1 SEQ ID NO: 4 GGGACCACTTTGTACAAGAAAGCTGGGTTTAAGCTCCGTG ATGGTGATGGTGATGTGCTC CATCATTCATGGTGAAGTC S1 F2 SEQID NO: 5 GGGACAAGTTTGTACAAAAA AGCAGGCTTCGAAGGAGATA GAACCATG F3 SEQ IDNO: 6 CAGGCTTCGAAGGAGATAGA ACCATGAAGGAAAATGTGGC ATCTGCAACC F4 SEQ ID NO:7 GAAGGAAAATGTGGCATCTG CAACCGTTTTCACTCTGCTA CTTTTTCTC F5 SEQ ID NO: 8CGTTTTCACTCTGCTACTTT TTCTCAACACCTGCCTTCTG AATGGAGGAG F6 SEQ ID NO: 9CAACACCTGCCTTCTGAATG GAGGAGCACATCACCATCAC CATCACGGAG F7 SEQ ID NO: 10CACATCACCATCACCATCAC GGAGCTCAGTTACCTCCTGG AAAACCTGAG R2 SEQ ID NO: 11GGGACCACTTTGTACAAGAA AGCTGGGTTCACTGAACTAT GTAAGTCACGTCCAC S2 F8 SEQ IDNO: 12 GGGACAAGTTTGTACAAAAA AGCAGGCTTCGAAGGAGATA GAACCATG F9 SEQ ID NO:13 CAGGCTTCGAAGGAGATAGA ACCATGAAGGAAAATGTGGC ATCTGCAACC F10 SEQ ID NO:14 GAAGGAAAATGTGGCATCTG CAACCGTTTTCACTCTGCTA CTTTTTCTC F11 SEQ ID NO: 15CGTTTTCACTCTGCTACTTT TTCTCAACACCTGCCTTCTG AATGTTCA F12 SEQ ID NO: 16TCTCAACACCTGCCTTCTGA ATGTTCAGCCAGACCCTCCT TTGGAGCTG R3 SEQ ID NO: 17CGTGATGGTGATGGTGATGT GCTCCATCATTCATGGTGAA GTCACTAGG R4 SEQ ID NO: 18CAAGAAAGCTGGGTTTAAGC TCCGTGATGGTGATGGTGAT GTGCTCC R5 SEQ ID NO: 19GGGACCACTTTGTACAAGAA AGCTGGGTTTAAGCTCC

For cloning of S1 and S2 domains, a nested PCR approach was adopted toincorporate tags and to engineer the 3′/5′ region. For S1, there are 6forward nested primers and 1 reverse primer for cloning. For S2, thereare 5 forward nested primers and 3 reverse primers for cloning.

PCR amplification was carried out using PfuUltra™ Hotstart PCR MasterMix (Stratagene) according to manufacturer's recommendation. Templateused for amplification is PRLR ECD fragment cloned in pDEST3218 (datanot shown). The ECD PCR product is cloned into BlueBac4.5/V5-His TOPO-TA(Invitrogen) using the topoisomerase cloning strategy. The S1 and S2 PCRproducts are cloned using Gateway Technology (Invitrogen) into in-houseadapted pAcMP3. The final selected clones were confirmed bydouble-strand sequencing. 10-20 ug of DNAs was prepared for insecttransfection.

The recombinant constructs were used to express the respective PRLRfragments in insect cells as follows. Baculovirus was isolated by plaquepurification of a co-transfection of plasmid DNA encoding theextracellular domain of PRLR with Sapphire™ genomic Autographacalifornica DNA. Recombinant virus was amplified and used to infect Tn5insect cells at densities ranging from 1×10⁶-1.5×10⁶ cells per ml, moirange of 2-10 in a 10 L (working volume) wavebioreactor. Following 48hour infection, cells and supernatant were collected, centrifuged andthe supernatant prepared for concentration. Supernatant was clarified ona 0.45 um hollow fiber cartridge before 5× concentration with tangentialflow 10 kDa MW cut-off membrane. Prior to protein purification,supernatant was filter sterilized w/1 L, 0.2 um pore vacuum flasks.

Similar methods were used to express the S1 and S2 domains in insectcells, except that S1 fragment was not concentrated before purificationand S2 fragment was concentrated on a 5 kDa MW cartridge.

PRLR fragments were purified as follows. Insect cell-culturesupernatants containing expressed PRLR ECD or subdomains was receivedfrom the expression group neat or concentrated up to 10× using a stackedmembrane cassette apparatus (Pall Filtron) with a 1 or 5 kD nominalmolecular weight cut-off. When practical, supernatants were filteredthrough a 0.2 micron filter. Supernatants were loaded directly ontoPBS-equilibrated columns.

His-tagged proteins were purified on 1- or 5-mL HisTrap columns (GEHealthcare) at the manufacturer's recommended flow rates. Glu-taggedprotein was purified on an immobilized anti-glu monoclonal antibodycolumn prepared as follows: purified anti-glu monoclonal antibody inPBS, at concentrations from 3-10 mg/mL, was conjugated to Affi-gel 10(Bio-Rad), an n-hydroxysuccinimide activated agarose gel, permanufacturer's instructions. Anti-glu agarose was packed into an XK 16column (GE Healthcare) and run at a linear flow rate of 15-30 cm/hr.

Elution of protein of the HisTrap columns was by a 20 column volumegradient elution from buffer A (PBS) to buffer B (PBS+0.25 M imidazole(IX-0005, EM Merck) pH 7.4). Elution of protein from the anti-glu columnwas by PBS containing 0.1 mg/mL of the peptide EYMPTD, which competeswith the Glu-Glu epitope. Fractions are examined by SDS-PAGE and Westernblot or mass spectrometry and pooled appropriately.

Pooled PRLR ECD or subdomains were further purified by size exclusionchromatography using a Superdex 75 26/60 column (GE Healthcare)equilibrated in PBS and run at 2.5 mL/min. No more than 10 mL was loadedonto these columns. Fractions were examined by SDS-PAGE and pooledappropriately.

Example 2 Isolation of Target-Specific Antibodies from Human AntibodyPhage Display Libraries

To isolate a panel of antibodies able to neutralize the activity ofhuman PRLR, three human antibody phage display libraries, expressingscFv fragments, were investigated in parallel. The target used for thelibrary panning was the soluble extracellular domain (ECD) of theprolactin receptor (human prolactin receptor amino acids 25-234)prepared as described above in Example 1. The receptor was biotinylated(NHS-LC biotin, Pierce) and soluble panning was performed on thebiotinylated ECD.

Selection of target specific antibody from phage display was carried outaccording to methods described by Marks et al. (Methods Mol. Biol.248:161-76, 2004). Briefly, the phage display library was incubated with50 pmols of the biotinylated ECD at room temperature for 1 hr and thecomplex formed was then captured using 100 μl of Streptavidin beadssuspension (Dynabeads® M-280 Streptavidin, Invitrogen). Non specificphages were removed by washing the beads with wash buffer (PBS+5% Milk).Bound phages were eluted with 0.5 ml of 100 nM Triethylamine (TEA) andimmediately neutralized by addition of an equal volume of 1M TRIS-Cl pH7.4. Eluted phage pool was used to infect TG1 E. coli cells growing inlogarithmic phase, and phagemid was rescued as described (Methods Mol.Biol. 248:161-76, 2004). Selection was repeated for a total of threerounds. Single colonies obtained from TG1 cells infected with elutedphage from the third round of panning were screened for binding activityin an ELISA assay. Briefly, single colonies obtained from the TG1 cellinfected with eluted phage were used to inoculate media in 96-wellplates. Microcultures were grown to an OD₆₀₀=0.6 at which pointexpression of soluble antibody fragment was induced by addition of 1 mMIPTG following overnight culture in a shaker incubator at 30° C.Bacteria were spun down and periplasmic extract was prepared and used todetect antibody binding activity to ECD immobilized on 96-wellmicroplates (96-well flat bottom Immunosorb plates, Nunc) followingstandard ELISA protocol provided by the microplate manufacturer.

The affinities of the anti-Prolactin Receptor (PRLR) antibodies forbinding to the recombinant extracellular domain (ECD) were estimatedusing the Biacore® 2000 and used for affinity ranking of antibodies. AProtein A/G capture surface was used for the human scFv-Fc fusions and arabbit anti-mouse IgG-Fc (RAM-Fc) antibody capture surface was used forthe antibodies produced by hybridomas. Both the Protein A/G and theRAM-Fc capture chips were CM5 sensor chips with maximal levels ofcapture molecule (either Protein A/G or RAM-Fc) immobilized on all fourflow cells via standard EDC-NHS amine coupling chemistries according tothe recommended protocol from Biacore® Inc. The running buffer wasHBS-EP (Biacore®, Inc.), the temperature was set at 25° C., and the flowrate was initially 10 μL/min. Purified antibodies were diluted intoHBS-EP to approximate concentrations between 1-3 μg/mL, and injectedover the capture chips for 1 to 2 minutes. The flow rate was increasedto 25 to 30 μL/min. The recombinant ECD of PRLR was diluted to 1 μg/mLand injected for 5 to 6 minutes with a 10 minute dissociation.

Fits were performed using BIAEvaluation software and used to calculatekinetic association and dissociation rate constants (k_(on) and k_(off),respectively). The 1:1 Langmuir interaction model with mass transportcorrection was used to perform the simultaneous ka/kd fit on eachsample. Several samples were fit at the same time with the Rmax, k_(on),and k_(off) parameters set to fit local. When baseline drift occurred,the drifting baseline model was used with the drift value set toconstant and entered manually. Drift values varied from −0.03 to +0.05RU/second.

Antibody binding was also assessed by measuring binding to prolactinreceptor expressing cells using Fluorescent Activated Cell Sorting(FACS) analysis and Fluorometric Microvolume Assay Technology (FMAT)(Swartzman et al., Anal Biochem. 271:143-51, 1999). Clones that showedbinding by either FACS or FMAT assays were sequence analyzed, and clonesencoding unique heavy chain CDR3 and light chain CDR3 protein sequenceswere reformatted to scFv-Fc as described in Example 3 below. ThesescFv-Fc were tested for ability to inhibit PRLR-induced ERK1/2phosphorylation and PRLR-induced proliferation of a BaF3/PRLR cell line,as described in Examples 5 and 6 below. Selected antibodies were furthercharacterized for binding to the ECD, S1 and S2, as well as for relativecompetition between pairs of antibodies for binding to PRLR ECD, asdescribed in Example 7. Data for selected antibodies is displayed belowin Table 4

TABLE 4 Epitope bin Affinity or (antibodies Proliferation Equilibrium insame bin pERK1/2 inhibition Dissociation compete inhibition IC50 forConstant Domain for binding Antibody IC50 BaF3/PRLR K_(D) (nM)specificity to PRLR) XPA.06.158 0.01 0.06 0.7 S1 4.5 XPA.06.167 0.040.14 4 S1 3.8 XPA.06.178 0.09 0.23 20 S1 3.8 XPA.06.145 0.30 0.77 10 S14 XPA.06.217 0.35 1.18 7 S1 7 XHA.06.983 0.11 0.1 0.1 S? 6 XHA.06.1890.2 0.15 <0.1 S1 3.8 XHA.06.275 0.5 1.31 0.4 S2 5 XHA.06.567 0.65 7.060.8 S2 6.5

Example 3 Reformatting of Clones to scFv-Fc Format

For each unique scFv clone identified in Example 2, the cDNA encodingthe scFv fragment is amplified by PCR from the phage display vector andligated into a mammalian expression vector, which is a modification ofXOMA's proprietary expression vector (described in WO 2004/033693,encoding either the kappa (κ), lambda (λ) or gamma-2 (γ2) constantregion genes), allowing expression of each antibody in an scFv-Fcprotein, where the Fc portion of the protein represents the CH2 and CH3domain of the IgG1 molecule. Construction of scFv-Fc fusion proteins iswell known in the art, for example see Fredericks et al, Protein Eng DesSel. 2004 January; 17(1):95-106, Powers et al, J Immunol Methods. 2001May 1; 251 (1-2):123-35, or Shu et al, Proc. Nat. Acad. Sci. USA 1993,90, 7995-7998. U.S. Pat. No. 5,892,019 also describes the constructionof the Fc fusion protein vector and the expression of scFv-Fc fusionproteins.

Expression of the fusion protein is performed by transfection of 293Esuspension cells with Lipofectamine 2000 (Invitrogen), using themanufacturer's instructions. After five days, the cells are removed bycentrifugation and the scFv-Fc fusion is purified from the supernatantusing protein A sepharose (GE Healthcare) using the manufacturer'ssuggested protocol.

Example 4 Identification of Target-Specific Antibodies Secreted byMurine Hybridomas

Mouse antibodies against the extra-cellular domain (ECD) of the humanProlactin receptor (PRLR) were generated as follows. Six Balb/C micewere immunized via subcutaneous injection with recombinant PRLRextra-cellular domain (described above). The mice received 10 injectionsover a 28 day period. Four days after the final injection the mice weresacrificed and the draining lymph nodes were harvested. After suspendingcells from the lymph nodes they were fused with the mouse myeloma cellline P3xAg8.653 by electrocell fusion using a BTX ECM2001 Electro-CellManipulator (Harvard Apparatus).

Following the fusion the cells were plated out into approximately 4096-well plates. After 12 days the plates were screened by ELISA againstthe recombinant ECD and in an FMAT. The FMAT assay used a CHO cell linestably transfected to express a high level of PRLR receptor.

Selected hybridomas were tested for ability to inhibit PRLR-inducedERK1/2 phosphorylation and PRLR-induced proliferation of a BaF3/PRLRcell line, as described in Examples 5 and 6 below. Selected antibodieswere further characterized for binding to recombinant ECD, S1 and S2, aswell as for relative competition between pairs of antibodies for bindingto PRLR ECD as described in Example 7. Data for selected antibodies isdisplayed above in Table 4.

Example 5 Determination of Antibody Effect on ERK1/2 Phosphorylation

Following a 5 hour serum starvation, T47D cells were seeded inmicrotiter plates in complete growth medium for 24 hours at 37° C. Cellswere washed twice with phosphate buffered saline (PBS) and incubatedwith antibodies diluted in serum-free media containing 0.1% BSA for 30minutes at 37° C. The final starting concentration of the antibodies was40 ug/ml. Media was removed and prolactin diluted in serum-free mediacontaining 0.1% BSA was added to a final concentration of 30 ng/ml.Cells were incubated with prolactin for 30 minutes at 37° C. followed bytwo washes with ice cold PBS. Standard lysis buffer containingdetergents, chelators, and various protease and phosphatase inhibitorswas added to generate cell lysates. The levels of phosphorylated ERK1/2(pERK1/2) were measured using standard ELISA according to instructionsof DUOSET® IC Phospho-ERK1/ERK2, R&D Systems, Inc. Results of arepresentative assay are displayed in FIGS. 2, 3 and 4 and results ofassay for selected antibodies are shown above in Table 4.

FIG. 7A-7C shows the VH and VL amino acid sequences of antibodies thathad greater than 80% inhibition in the pERK assay.

Example 6 Determination of Antibody Effect on Proliferation ofPRL-Responsive Cell Lines

BaF3/PRLR cells were generated by electroporating the murine pro-B cellline BaF3 with an expression vector containing the full-length humanPRLR and a neomycin resistance cassette. Cells were selected for 7 daysin media supplemented with G418 (1 mg/ml) and rmIL-3 (10 ng/ml),followed by a 7 day selection period in rhPRL (1 ug/ml) without G418 orIL-3. Over a 14 day period, the media PRL concentration was reducedstepwise until a maintenance level of 50 ng/ml was reached. On the dayof the experiment, 1×10⁴ cells were seeded into each well of a flatbottom 96 well plate. Antibodies (in scFv-Fc fusion format) were addedto wells at a concentration of 10 ug/ml, with and without 50 ng/mlrhPRL. Plates were incubated for 48 hr and analyzed using CellTiter Gloreagent. Samples were run in triplicate, agonism was assessed by cellproliferation induced by antibodies in the absence of PRL whileantagonism was determined by cell proliferation in the presence of PRL.Results of this proliferation assay for selected antibodies are shownabove in Table 4.

In order to analyze PRL-induced proliferation and inhibition ofproliferation by anti-PRLR antibodies, T47D or MCF7-NCl cells were splitat a density of 1×10⁶ cells per ml of regular growth media (phenolred-free RPMI/10% FCS) into a T75 flask (12 ml total volume). 72 hrsafter split, cells were trypsinized, counted, and seeded at a density of5K per well (T47D) or 20K per well (MCF7) of a flat bottom 96 well plate(100 ul per well). MCF7 cells were seeded in serum-free and phenolred-free RPM1, T47D cells were seeded in either serum-free RPMI or RPMIcontaining 10% charcoal-stripped serum. 24 hrs after seeding, PRL andanti-PRLR antibodies were added to wells (50 ul, 3× concentrated). After72 hrs of incubation, ³[H] thymidine (1 μci per well) was added to theplate for a minimum of 6 hrs in a 37° incubator. Cells were harvestedusing trypsin and a Tomtec 96 well plate cell harvester. Filters werethen transferred to a Trilux luminometer and analyzed (1 min counts).Results of the proliferation study in FIG. 5 show that scFv inhibits theprolactin-mediated increase in proliferation.

Example 7 Measurement of Binding Affinity and Competition via BIACORE

BIACORE analysis as described above in Example 2 was repeated todetermine relative binding of selected antibodies to ECD, S1 and S2domains of PRLR as described above, except that the St, S2, or ECDproteins were injected at 10 μg/mL for 2 minutes at 15 μL/minute. Datafrom this assay were collected as report points (resonance units (RU))by the Biacore® control software, and normalized by dividing the amountof antigen bound by the amount of antibody captured. Data are shown inTable 5 below.

TABLE 5 Normalized S1, S2 and ECD binding by anti-PRLR antibodies. PRLRFragment Bound (RU bound/RU Ab Captured) Sample S1 S2 ECD XPA.06.1453.0% −0.8% 4.8% XPA.06.158 20.5% 0.1% 28.2% XPA.06.167 23.7% 0.1% 35.0%XPA.06.178 16.4% 0.0% 20.5% XPA.06.217 19.4% 0.4% 25.8% XHA.06.567 0.9%16.7% 31.1% XHA.06.983 −2.2% −2.5% 27.4% XHA.06.275 0.0% 13.4% 26.2%XHA.06.189 16.9% 0.2% 31.6%

The affinities of the purified antibodies were determined by performinga series of injections on the Biacore® 2000. The affinity and rateconstants generated are relevant for these antibodies binding therecombinant extra-cellular domain (ECD) of the prolactin receptor (PRLR)at 25° C. in an HBS-EP buffer system. A CM5 sensor chip withapproximately 5000-1000 RU of Protein A/G was prepared via standardEDC-NHS amine coupling chemistries according to the recommended protocolfrom Biacore® Inc. and used to capture the antibodies. The purifiedantibodies were diluted to roughly 1 ug/mL in HBS-EP buffer for capture.Injection time required to give between 250 and 400 RU of antibodycapture was determined. The capture of the antibodies for the kineticanalysis was performed by injecting the antibodies at 10 uL/minute for1.5 to 3 minutes, depending on results of the capture leveloptimization.

For the kinetic analysis, the flow rate was set at 40 uL/minute. Fiveconcentrations of PRLR ECD were prepared in a 1:3 serial dilution fromeither 148 nM (4 ug/mL) or 37 nM (1 ug/mL). Each concentration plus abuffer control (zero concentration) were injected in duplicate. The datasets were double referenced and fit globally using a 1:1 Langmuirbinding interaction model. This same analysis was also performed for theIgG reformatted construct of XPA.06.167 for both IgG1 and IgG2constructs.

Kinetic constants and affinities for binding of selected antibodies toECD of PRLR is displayed in Table 6 below.

TABLE 6 Affinity analysis results ANTIBODY k_(on) (1/Ms) k_(off) (1/s)K_(D) (M) XPA.06.131 3.4E+04 7.3E−05 2.1E−09 XPA.06.158 1.1E+05 7.3E−057.0E−10 XPA.06.141 6.3E+04 5.3E−04 8.4E−09 XPA.06.147 5.5E+05 5.3E−039.8E−09 XPA.06.167 IgG1 2.3E+05 6.0E−04 2.6E−09 XPA.06.167 IgG2 2.1 + 055.72E−04  2.7E−09

Similar procedures were used to determine kinetic constants andaffinities for additional antibodies (Summarized in Table 7, below).

TABLE 7 kon koff KD XHA.06.642 9.2E+05 8.6E−04 934 pM XHA.06.275 1.0E+063.4E−04 337 pM XHA.06.983 7.3E+05 3.2E−04  43 pM chXHA.06.642 6.5E+045.2E−04 801 pM chXHA.06.275 1.1E+06 2.2E−04 196 pM chXHA.06.983 2.4E+051.0E−05  42 pM

Relative competition or interference between pairs of antibodies (e.g.,pairing analysis) for binding to PRLR was determined as follows in aserial competition assay strategy. In this approach, one antibody isimmobilized onto a sensor chip, either directly or through a captureagent, and allowed to bind the ECD as it is injected over theimmobilized antibody. When necessary, excess captured agent is blockedby injecting a high concentration of irrelevant IgG (e.g., when testingtwo murine antibodies using a rabbit-anti-mouse IgG capture surface).The antibody to be tested for competition is subsequently injected, andits ability to bind the ECD captured by the first antibody isdetermined. If the two antibodies bind to spatially separated epitopeson the ECD, then the second antibody should also be able to bind theECD/first antibody complex. If the two antibodies interfere or compete,then the second antibody will not be able to bind as well, or at all, tothe ECD/first antibody complex. Results of this competition analysis aredisplayed in Table 4 above (if two antibodies have the same epitope binnumber, they will compete with each other for binding to ECD and theywill exhibit the same pattern of competition against antibodies fromother bins). The invention specifically contemplates the identificationof other antibodies that bind to the same epitope of PRLR as any of theantibodies in the bins described herein or that compete with suchantibodies for binding PRLR ECD.

Example 8 Effect on PRL-Induced PRLR, STAT5 & AKT Phosphorylation byWestern Blot

The ability of selected antibodies to inhibit PRL-inducedphosphorylation of STAT5 and AKT was determined as follows. Cells wereseeded overnight in 6 well plates at a density of 3×10⁵ cells/ml inphenol red-free RPMI/10% FBS. The following day, media was replaced withserum-free RPMI for 30 min. In some experiments, anti-PRLR ornon-specific control antibodies were incubated with cells during thisserum starvation period. 50 ng/ml PRL was then added to wells for 30min, after which cells were rinsed once in PBS and lysed in a bufferconsisting of 50 mM Tris-HCl, pH7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1mM Na3OV4, 50 mM NaF, 0.25% deoxycholate and protease inhibitors. Tubeswere spun down at 14,000 rpm in a refrigerated microfuge and lysateswere quantitated using BCA reagents. 30 μg of whole cell lysate wasseparated by 10% SDS-PAGE and proteins were detected usingphospho-specific antibodies for STAT5A/B (Y694/Y699, Upstate) or PRLR(Y546/Y611, in-house) and ECL. Equal protein loading was determined bystaining with antibodies specific for total STAT5 (BD) or PRLR (Zymed)or AKT (Cell Signalling). Results of a representative assay of effect ofa PRLR-specific antibody on PRLR intracellular phosphorylation are shownin FIG. 6.

Example 9 Humanization of Murine Antibodies

This example sets out a procedure for humanization of a murine anti-PRLRantibody.

Design of Genes for Humanized PRLR Antibody Light and Heavy Chains

The VL and VH amino acid sequences for murine antibodies XHA.06.983,XHA.06.275, and XHA.06.642 are set forth in FIG. 10. The sequence of ahuman antibody identified using the National Biomedical FoundationProtein Identification Resource or similar database is used to providethe framework of the humanized antibody. To select the sequence of thehumanized heavy chain, the murine heavy chain sequence is aligned withthe sequence of the human antibody heavy chain. At each position, thehuman antibody amino acid is selected for the humanized sequence, unlessthat position falls in any one of four categories defined below, inwhich case the murine amino acid is selected:

(1) The position falls within a complementarity determining region(CDR), as defined by Kabat, J. Immunol., 125, 961-969 (1980);

(2) The human antibody amino acid is rare for human heavy chains at thatposition, whereas the murine amino acid is common for human heavy chainsat that position;

(3) The position is immediately adjacent to a CDR in the amino acidsequence of the murine heavy chain; or

(4) 3-dimensional modeling of the murine antibody suggests that theamino acid is physically close to the antigen binding region.

To select the sequence of the humanized light chain, the murine lightchain sequence is aligned with the sequence of the human antibody lightchain. The human antibody amino acid is selected at each position forthe humanized sequence, unless the position again falls into one of thecategories described above and repeated below:

(1) CDR's;

(2) murine amino acid more typical than human antibody;

(3) Adjacent to CDR's; or

(4) Possible 3-dimensional proximity to binding region.

The actual nucleotide sequence of the heavy and light chain genes isselected as follows:

(1) The nucleotide sequences code for the amino acid sequences chosen asdescribed above;

(2) 5′ of these coding sequences, the nucleotide sequences code for aleader (signal) sequence. These leader sequences were chosen as typicalof antibodies;

(3) 3′ of the coding sequences, the nucleotide sequences are thesequences that follow the mouse light chain J5 segment and the mouseheavy chain J2 segment, which are part of the murine sequence. Thesesequences are included because they contain splice donor signals; and

(4) At each end of the sequence is an Xba I site to allow cutting at theXba I sites and cloning into the Xba I site of a vector.

Construction of Humanized Light and Heavy Chain Genes

To synthesize the heavy chain, four oligonucleotides are synthesizedusing an Applied Biosystems 380B DNA synthesizer. Two of theoligonucleotides are part of each strand of the heavy chain, and eacholigonucleotide overlaps the next one by about 20 nucleotides to allowannealing. Together, the oligonucleotides cover the entire humanizedheavy chain variable region with a few extra nucleotides at each end toallow cutting at the Xba I sites. The oligonucleotides are purified frompolyacrylamide gels.

Each oligonucleotide is phosphorylated using ATP and T4 polynucleotidekinase by standard procedures (Maniatis et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1989)). To anneal the phosphorylated oligonucleotides,they are suspended together in 40 ul of TA (33 mM Tris acetate, pH 7.9,66 mM potassium acetate, 10 mM magnesium acetate) at a concentration ofabout 3.75 μM each, heated to 95° C. for 4 min. and cooled slowly to 4°C. To synthesize the complete gene from the oligonucleotides bysynthesizing the opposite strand of each oligonucleotide, the followingcomponents are added in a final volume of 100 ul:

10 ul annealed oligonucleotides 0.16 mM each deoxyribonucleotide 0.5 mMATP 0.5 mM DTT 100 ug/ml BSA 3.5 ug/ml T4 g43 protein (DNA polymerase)25 ug/ml T4 g44/62 protein (polymerase accessory protein) 25 ug/ml 45protein (polymerase accessory protein)

The mixture is incubated at 37° C. for 30 min. Then 10 u of T4 DNAligase is added and incubation at 37° C. is resumed for 30 min. Thepolymerase and ligase are inactivated by incubation of the reaction at70° C. for 15 min. To digest the gene with Xba I, 50 ul of 2×TAcontaining BSA at 200 ug/ml and DTT at 1 mM, 43 ul of water, and 50 u ofXba I in 5 ul is added to the reaction. The reaction is incubated for 3hr at 37° C., and then purified on a gel. The Xba I fragment is purifiedfrom a gel and cloned into the Xba I site of the plasmid pUC19 bystandard methods. Plasmids are purified using standard techniques andsequenced using the dideoxy method.

Construction of plasmids to express humanized light and heavy chains isaccomplished by isolating the light and heavy chain Xba I fragments fromthe pUC19 plasmid in which it had been inserted and then inserting itinto the Xba I site of an appropriate expression vector which willexpress high levels of a complete heavy chain when transfected into anappropriate host cell.

Synthesis and Affinity of Humanized Antibody

The expression vectors are transfected into mouse Sp2/0 cells, and cellsthat integrate the plasmids are selected on the basis of the selectablemarker(s) conferred by the expression vectors by standard methods. Toverify that these cells secreted antibody that binds to PRLR,supernatant from the cells are incubated with cells that are known toexpress PRLR. After washing, the cells are incubated withfluorescein-conjugated goat anti-human antibody, washed, and analyzedfor fluorescence on a FACSCAN cytofluorometer.

The cells producing the humanized antibody are cultured in vitro.Humanized antibody is purified to substantial homogeneity from the cellsupernatants by passage through an affinity column of Protein A(Pro-Chem. Inc., Littleton, Mass. or equivalent) according to standardtechniques. The affinity of the humanized antibody relative to theoriginal murine antibody is determined according to techniques known inthe art.

Example 10 Human Engineering™ of Murine Antibodies

This example describes cloning and expression of Human Engineered™antibodies, as well as purification of such antibodies and testing forbinding activity.

Design of Human Engineered™ Sequences

Human Engineering™ of antibody variable domains has been described byStudnicka [See, e.g., Studnicka et al. U.S. Pat. No. 5,766,886;Studnicka et al. Protein Engineering 7: 805-814 (1994)] as a method forreducing immunogenicity while maintaining binding activity of antibodymolecules. According to the method, each variable region amino acid hasbeen assigned a risk of substitution. Amino acid substitutions aredistinguished by one of three risk categories: (1) low risk changes arethose that have the greatest potential for reducing immunogenicity withthe least chance of disrupting antigen binding; (2) moderate riskchanges are those that would further reduce immunogenicity, but have agreater chance of affecting antigen binding or protein folding; (3) highrisk residues are those that are important for binding or formaintaining antibody structure and carry the highest risk that antigenbinding or protein folding will be affected. Due to thethree-dimensional structural role of prolines, modifications at prolinesare generally considered to be at least moderate risk changes, even ifthe position is typically a low risk position. FIG. 10 shows the lightand heavy chain variable region amino acid sequences of murineantibodies XHA.06.983, XHA.06.275, and XHA.06.642.

Variable regions of the light and heavy chains of the murine antibodiesare Human Engineered™ using this method. Amino acid residues that arecandidates for modification according to the method at low riskpositions are identified by aligning the amino acid sequences of themurine variable regions with a human variable region sequence. Any humanvariable region can be used, including an individual VH or VL sequenceor a human consensus VH or VL sequence. The amino acid residues at anynumber of the low risk positions, or at all of the low risk positions,can be changed.

Similarly, amino acid residues that are candidates for modificationaccording to the method at all of the low and moderate risk positionsare identified by aligning the amino acid sequences of the murinevariable regions with a human variable region sequence. The amino acidresidues at any number of the low or moderate risk positions, or at allof the low and moderate risk positions, can be changed.

Preparation of Human Engineered™ Antibody Sequences

DNA fragments encoding Human Engineered™ heavy and light chain V regionsequences along with signal sequences (e.g., antibody-derived signalsequences) are constructed using synthetic nucleotide synthesis. DNAencoding each of the light chain V region amino acid sequences describedherein is inserted into a vector containing the human Kappa or Lambdalight chain constant region. DNA encoding each of the heavy chain Vregion amino acid sequences described herein is inserted into a vectorcontaining the human Gamma-1, 2, 3 or 4 heavy chain constant region. Allof these vectors contain a promoter (e.g., hCMV promoter) and a 3′untranslated region (e.g., mouse kappa light chain 3′ untranslatedregion) along with additional regulatory sequences, depending on theiruse for transient expression or stable cell line development (US2006/0121604).

For expression of Human Engineered™ antibodies using the aforementionedvectors containing variable region sequences, at least four variants maybe generated from different combinations of low risk light chain,low+moderate risk light chain, low risk heavy chain, and low+moderaterisk heavy chain. In those instances when moderate risk changes are notincluded in either or both of the light chain or heavy chain, fewervariants are correspondingly produced.

Preparation of Expression Vectors for Transient Expression

Vectors containing either the light or heavy chain genes described aboveare constructed for transient transfection. In addition to the HumanEngineered™ antibody sequences, promoter and light chain 3′ untranslatedregion described above, these vectors preferably contain theEpstein-Barr virus oriP for replication in HEK293 cells that express theEpstein-Barr virus nuclear antigen.

Transient Expression of Human-Engineered™ PRLR antibody in HEK293E Cells

Separate vectors each containing oriP from the Epstein-Barr virus andthe light chain or heavy chain genes described above are transfectedtransiently into HEK293E cells as described in US 2006/0121604.Transiently transfected cells are allowed to incubate for up to 10 daysafter which the supernatant is recovered and antibody purified usingProtein A chromatography.

Preparation of Expression Vectors for Permanent Cell Line Development

In addition to the Human Engineered™ antibody sequences, promoter andlight chain 3′ untranslated region described above, vectors forpermanent cell line development contain the selectable marker genes suchas neo or his for selection of G418—or histidinol—resistanttransfectants, respectively. A final vector is constructed that containsone copy of the heavy chain and one copy of the light chain codingregions.

Development of Permanently Transfected CHO-K1 Cells

The vectors described above containing one copy each of the light andheavy genes together are transfected into Ex-Cell 302-adapted CHO-K1cells. CHO-K1 cells adapted to suspension growth in Ex-Cell 302 mediumare typically transfected with linearized vector using linearpolyethyleneimine (PEI). The cells are plated in 96 well platescontaining Ex-Cell 302 medium supplemented with 1% FBS and G418. Clonesare screened in 96 well plates and the top ˜10% of clones from eachtransfection are transferred to deep-well 96 well plates containingEx-Cell 302 medium supplemented with G418.

A productivity test is performed in deep-well 96 well plates in Ex-Cell302 medium for cultures grown for 14 days at which time culturesupernatants are tested for levels of secreted antibody by animmunoglobulin ELISA assay for IgG.

The top clones are transferred to shake flasks containing Ex-Cell 302medium. Shake flask tests are performed with these clones in Ex-Cell 302medium. The cells are grown for 14 days in 125 ml Erlenmeyer flaskscontaining 25 ml media. The levels of immunoglobulin polypeptide in theculture medium are determined by IgG ELISA or HPLC at the end of theincubation period. Multiple sequential transfections of the same cellline with two or three multi-unit transcription vectors results inclones and cell lines that exhibit further increases in levels ofimmunoglobulin production, preferably to 300 μg/ml or more.

Purification

A process for the purification of immunoglobulin polypeptides fromvectors and all lines according to the invention may be designed (See,for example, US 2006/0121604). For example, according to methods wellknown in the art, cells are removed by filtration after termination. Thefiltrate is loaded onto a Protein A column (in multiple passes, ifneeded). The column is washed and then the expressed and secretedimmunoglobulin polypeptides are eluted from the column. For preparationof antibody product, the Protein A pool is held at a low pH (pH 3 for aminimum of 30 minutes and a maximum of one hour) as a viral inactivationstep. An adsorptive cation exchange step is next used to further purifythe product. The eluate from the adsorptive separation column is passedthrough a virus retaining filter to provide further clearance ofpotential viral particles. The filtrate is further purified by passingthrough an anion exchange column in which the product does not bind.Finally, the purification process is concluded by transferring theproduct into the formulation buffer through diafiltration. The retentateis adjusted to a protein concentration of at least 1 mg/mL and astabilizer is added.

Binding Activity

The PRLR binding activity of the recombinant Human Engineered™antibodies is evaluated. Protein is purified from shake flask culturesupernatants by passage over a protein A column followed byconcentration determination by A₂₈₀. Binding assays are performed asdescribed in other examples.

Example 11 Human Engineered Antibodies

Three of the aforementioned murine antibodies were Human Engineered™generally as described in Example 10.

Human Engineering™ of the Prolactin Receptor Antibodies XHA.06.642 andXHA.06.275

For XHA.06.642, the heavy chain was Human Engineered™ at either 11 lowrisk or 13 low plus moderate risk positions; the light chain was HumanEngineered™ only at low risk positions (14 changes) because all of themoderate risk positions already were human amino acids. For XHA.06.275,the heavy chain was Human Engineered™ at either 7 low risk or 11 lowplus moderate risk positions; the light chain was Human Engineered™ ateither 8 low risk or 10 low plus moderate risk positions.

Amino acid sequences of the Human-Engineered™ variable regions derivedfrom XHA.06.642, XHA.06.275 and XHA.06.983 are shown below (CDRsunderlined). These variable regions were assembeled in variouscombinations (e.g., SEQ ID NO: 88 and SEQ ID NO: 89; SEQ ID NO: 88 andSEQ ID NO: 90; SEQ ID NO: 91 and SEQ ID NO: 93; SEQ ID NO: 91 and SEQ IDNO: 94; SEQ ID NO: 92 and SEQ ID NO: 93; or SEQ ID NO: 92 and SEQ ID NO:94) to generate the Human-Engineered™ antibodies he.06.642-1,he.06.642-2, he.06.275-1, he.06.275-2, he.06.275-3, he.06.275-4.

The CDRs of SEQ ID NO: 88 (he.06.642 light chain variable region lowrisk) underlined below are at positions 24 through 38, positions 54through 60, and positions 93 through 101 of the amino acid sequence ofSEQ ID NO: 88. The CDRs of SEQ ID NO: 89 (he.06.642 heavy chain variableregion low risk) underlined below are at positions 31 through 35,positions 50 through 66, and 99 through 113 of SEQ ID NO: 89. The CDRsof SEQ ID NO: 90 (he.06.642 heavy chain variable region low+moderaterisk) underlined below are at positions 31 through 35, positions 50through 66, and 99 through 113 of SEQ ID NO: 90.

he.06.642 LC Variable Region Low Risk (SEQ ID NO: 88):DIVLTQSPDSLAVSLGERATINCKASKSVSTSGYTYMHWYQQKPGQPPKLLIYLASNRESGVPDRFSGSGSGTDFTLTISPVQAEDVATYYCQHSGELPP SFGQGTKLEIK he.06.642HC Variable Region Low Risk (SEQ ID NO: 89):EVQLVESGGGLVQPGGSLRLSCAVSGFTFSSYGMSWVRQAPGKRLEWVATVSSGGTYTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAMYYCARHRGNYYATYYYAMDYWGQGTLVTVSS he.06.642 HC Variable Region Low + ModerateRisk (SEQ ID NO: 90): EVQLVESGGGLVQPGGSLRLSCAVSGFTFSSYGMSWVRQAPGKGLEWVATVSSGGTYTYYPDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHRGNYYATYYYAMDYWGQGTLVTVSS he.06.275 LC Variable Region Low Risk (SEQ IDNO: 91): DVQITQSPSSLSASPGDRITLTCRASKNIYKYLAWYQEKPGKTNNLLIYSGSTLHSGIPSRFSGSGSGTDFTLTISSLQPEDFAMYYCQQHNDYPYTFGQ GTKLEIK he.06.275 LCVariable Region Low + Moderate Risk (SEQ ID NO: 92):DVQITQSPSSLSASPGDRITLTCRASKNIYKYLAWYQEKPGKANKLLIYSGSTLHSGIPSRFSGSGSGTDFTLTISSLQPEDFAMYYCQQHNDYPYTFGQ GTKLEIK he.06.275 HCVariable Region Low Risk (SEQ ID NO: 93):DVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQFPGKKLEWMGYISYSGSTSYNPSLKSRITISRDTSKNQFSLQLNSVTAADTATYFCARDY GYVFDYWGQGTTLTVSShe.06.275 HC Variable Region Low + Moderate Risk (SEQ ID NO: 94):DVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQFPGKKLEWMGYISYSGSTSYNPSLKSRITISRDTSKNQFSLQLNSVTAADTATYFCARDY GYVFDYWGQGTTLTVSShe.06.983 LC Variable Region Low Risk (SEQ ID NO: 95):DIVMTQSPDSLAVSAGERVTINCKASQGVSNDVAWFQQKPGQSPKLLIYSASTRYTGVPDRLSGSGSGTDFTFTISSVQAEDVAVYFCQQDYTSPTFGQG TKLEIK he.06.983 LCVariable Region Low + Moderate Risk (SEQ ID NO: 96):DIVMTQSPDSLAVSAGERVTINCKASQGVSNDVAWFQQKPGQSPKLLIYSASTRESGVPDRLSGSGSGTDFTFTISSVQAEDVAVYFCQQDYTSPTFGQG TKLEIK he.06.983 HCVariable Region Low Risk (SEQ ID NO: 97):DVQLVESGGGLVQPGGSRRLSCAASGFAFSSFGMQWVRQAPGKGLEWVAYISSGSSTIYYADTVKGRFTISRDNPKNTLYLQMNSLRAEDTAMYYCVRSG RDYWGQGTLVTVSShe.06.983 HC Variable Region Low + Moderate Risk (SEQ ID NO: 98):EVQLVESGGGLVQPGGSRRLSCAASGFAFSSFGMQWVRQAPGKGLEWVAYISSGSSTIYYADSVKGRFTISRDNPKNTLYLQMNSLRAEDTAVYYCVRSG RDYWGQGTLVTVSS

Example 12 Expression and Purification of he.06.642 and he.06.275Antibodies

The Human Engineered™ he.06.642 and he.06.275 light and heavy chain Vregions were fused to human Kappa and Gamma-1 or Gamma-2 constantregions, respectively. The heavy and light chain genes then were fusedto a strong promoter and efficient 3′ untranslated region and clonedinto transient expression vectors containing the Epstein-Bar virusorigin of replication

Antibodies were transiently expressed in HEK293 cells using separateplasmids encoding antibody heavy chain and light chain sequences in thevarious Low or Low+Moderate Risk combinations generally as described inExample 10. The ratio of heavy chain:light chain DNA was 1:2.Transfection of cells was performed with PEI at a ratio of DNA:PEI of1:2 and a DNA concentration of 1 ug/mL. The cell density was 8e5cells/mL. The DNA was prepared using standard Qiagen kits. Theexpression cultures were grown in IS293 media (Irvine Scientific)+1% lowIg FBS (Hyclone) in 2 L flasks with 400 mL media per flask. Cultureconditions were 37° C., 5% CO₂, and agitation at 90-95 RPM. After 5-7days in culture, the culture medium was harvested and clarified as thepurification input.

Purification of chimeric and Human Engineered™ versions of theaforementioned antibodies was achieved in a single step by passingtransient expression culture supernatant directly over a recombinantProtein A Fast Flow column (GE Healthcare). Elution of the major peakwas by 0.1 mM glycine pH 3.5. Pooled material was dialyzed into PBS andconcentrated in centrifugal concentrators with a nominal molecularweight cut-off of 30 kD. Final purities were >95% and overall yieldswere approximately 60%. The final pools were assayed for endotoxin usingan Endosafe PTS LAL unit (Charles River) or the QCL-1000 Chromogenic LALEndpoint Assay (Lonza), and the results were <0.05 EU/mg (below thelimit of detection) for all the antibodies. Aggregation state of thechimeric antibody he.06.642-2 was determined to be monomeric by SEC on aSuperdex 200 10/300 GL column (GE Healthcare).

chXHA.06.642 is isolated to a purity of >95% in a single chromatographicstep followed by dialysis for buffer exchange. chXHA.06.642 is solublein PBS at 3 mg/mL and no major impurities or aggregation are detected asmeasured by size exclusion chromatography.

Testing of Human Engineered Anti-human PRLR Antibodies by Flow Cytometry

CHO-K1 parental and human prolactin receptor (PRLR) expressing cellswere harvested, centrifuged and resuspended at approximately 5×10⁶cells/ml in 1×PBS containing 2% FBS and 0.1% sodium azide (FACS buffer).Human engineered anti-human PRLR and anti-KLH isotype control antibodieswere diluted to 2× final concentration in FACS buffer and added toappropriate sample wells (50 ml/well). For secondary antibody andautofluorescence controls, 50 ml FACS buffer was added to appropriatewells. 50 ml of cell suspension was added to each sample well. Sampleswere incubated at 4° C. for 1 hour, washed 2× with cold FACS buffer andresuspended in FACS buffer containing PE-conjugated goat anti-human IgG(Jackson Immunoresearch, West Grove, Pa.) at a 1:100 dilution. Followinga 30 minute incubation at 4° C., cells were washed 2× with cold FACSbuffer, resuspended in FACS buffer containing 1 mg/ml propidium iodide(Invitrogen, San Diego, Calif.) and analyzed by flow cytometry. As shownin Table 7, the anti-PRLR antibodies bind to the PRLR expressing cellsbut not the parental cells.

TABLE 8 PLRL Cell Line, clone 1G5 CHO-K1 Parental Cell Line Sample MFI:Sample MFI: Auto. control, 1G5 2.51 Auto. control, 1G5 2.66 GAH-PE, 1G52.6 GAH-PE, PAR 2.54 GAM-PE, 1G5 2.61 GAM-PE, PAR 2.58 MAB1167, 1G5 33.8MAB1167, PAR 2.58 KLH8.G2, 1G5 2.75 KLH8.G2, PAR 2.66 he.06.642 3 G2,1G5 38.5 he.06.642-3 G2, PAR 2.58 KLH8.G1, 1G5 2.69 KLH8.G1, PAR 2.59he.06.642 3 G1 (1), 1G5 39.1 he.06.642 3 G1 (1), PAR 2.57 he.06.642 3 G1(2), 1G5 43 he.06.642 3 G1 (2), PAR 2.55 chXHA.06.642 (1), 1G5 37.3chXHA.06.642 (1), PAR 2.56 chXHA.06.642 (2), 1G5 37.3 chXHA.06.642 (2),PAR 2.55

Example 13 Affinity of Human Engineered™ Antibodies

Affinity of the chimerized and Human Engineered™ antibodies determinedby Biacore analysis. Briefly, a CM5 sensor chip (Biacore) immobilizedwith Protein A/G (Pierce) via NHS/EDC was used to capture approximately600 RU of antibody on the chip surface. Five concentrations of PRLR ECDbeginning at 5 ug/mL (185 nM) and serially diluted at 5× dilution to 0.3nM were injected from lowest to highest concentration in the kinetictitration injection mode, and 15 minutes of dissociation data wascollected. The experiments were double-referenced, i.e. an adjacent flowcell response was subtracted automatically, and the response from abuffer injection experiment was subtracted from the experimental dataset. Kinetic and derived parameters (k_(a), k_(d) and K_(D)) weredetermined by fitting to a 1:1 Langmuir model using BiaEval softwarecustomized to the kinetic titration injection mode. Affinitymeasurements of both chimeric and all Human Engineered™ antibodiesagainst human and cynomolgus PRLR ECD are summarized in Table 9 andTable 10.

TABLE 9 Sample KD kd ka Chi2 chXHA.06.275 Human 3.9E−10 2.7E−04 7.0E+050.574 he.06.275-1 Human 5.0E−10 3.1E−04 6.2E+05 0.594 he.06.275-2 Human6.1E−10 3.7E−04 6.0E+05 0.532 he.06.275-3 Human 4.3E−10 2.7E−04 6.4E+050.555 he.06.275-4 Human 5.2E−10 3.2E−04 6.2E+05 0.69 chXHA.06.275 Cyno1.2E−09 5.0E−04 4.3E+05 1.48 he.06.275-1 Cyno 1.6E−09 5.7E−04 3.6E+052.78 he.06.275-2 Cyno 1.9E−09 6.9E−04 3.7E+05 1.49 he.06.275-3 Cyno1.3E−09 5.0E−04 3.9E+05 1.19 he.06.275-4 Cyno 1.7E−09 6.0E−04 3.6E+051.2 chXHA.06.642 Human 1.3E−09 4.6E−04 3.5E+05 5.15 he.06.642-1 Human2.0E−09 3.5E−04 1.7E+05 12.9 he.06.642-2 Human 3.1E−09 5.3E−04 1.7E+0512.6 chXHA.06.642 Cyno  26E−08 4.3E−03 1.7E+05 12.6 he.06.642-1 Cyno3.1E−08 3.8E−03 1.2E+05 8.29 he.06.642-2 Cyno 4.7E−08 8.3E−03 1.8E+0511.1

TABLE 10 Sample ka kd KD Res SD he.06.642 3 -G1 lot1 1.038(1)e53.828(5)e−4 3.688(5) nM 0.977 he.06.642 3 -G1 lot2 1.02E+5 3.88E−43.79977 nM 1.06 he.06.642 3 -G2 9.801(1)e4 4.210(7)e−4 4.296(6) nM 1.054chXHA.06.642 1.962(3)e5 6.47E−04 3.296(5) nM 1.389 CHO.KLHG2-60 Nobinding No binding No binding No binding

In order to compare rat and mouse cross reactivity using covalentlyimmobilized antibodies, a CM4 chip was coupled with he.06.642-2 andhe.06.275-4 antibodies via standard EDC-NHS amine coupling chemistriesaccording to the recommended protocol from Biacore® Inc. The PRLR ECDinjections were performed at five concentrations in a three foldtitration series starting at 111 nM and going down to 1.37 nM.Regeneration was performed with Glycine pH3.0. The affinity of all ofthe HE variants of XHA.06.642 and XHA.06.275 are very similar to theaffinity of the parental chimeric antibody. Antibody he.06.642-2 bindsto human, mouse and rat PRLR with equivalent affinity. It binds to CynoPRLR with 15 fold weaker affinity than human PRLR. Antibody he.06.275-4binds to Cyno PRLR with 5 fold weaker affinity than Human PRLR. Antibodyhe.06.275-4 does not bind effectively to Mouse or Rat PRLR. A summary ofthe data is provided in Table 11.

TABLE 11 Cross Species Affinity Analysis of Select HE Variants onCovalently Immobilized Antibodies he.06.642-2 Results Sample kon koff KD(nM) he.06.642-2 Human 3.5E+05 9.1E−04 2.6 he.06.642-2 Cyno 1.5E+056.0E−03 38.9 he.06.642-2 Murin 1.1E+05 3.1E−04 2.7 he.06.642-2 Rat7.6E+04 1.4E−04 1.9 Cyno KD/Human KD = 15 Murine KD/Human KD = 1 RatKD/Human KD = 0.75 he.06.275-4 Results Sample kon koff KD (nM)he.06.275-4 Human 3.4E+05 4.5E−04 1.3 he.06.275-4 Cyno 1.3E+05 8.2E−046.4 he.06.275-4 Murin 2.1E+03 3.7E−02 17,613 he.06.275-4 Rat 0.0E+000.00E−00  0 Cyno KD/Human KD = 5 Murine KD/Human KD = 13,548 RatKD/Human KD =

Example 14 Inhibition of BaF/PRLR Cell Proliferation and Survival andInhibition of PRLR-induced ERK1/2 Phosphorylation

Chimeric mAbs were analyzed for their ability to inhibit theproliferation and survival of BaF/PRLR cells [FIG. 11]. All chimerastested were found to have retained their potencies relative to theircorresponding hybridoma clones. In fact, XHA.06.642 and XHA.06.275 werefound to have gained potency in this assay following chimerization. Inorder to assess the PRLR signal-neutralizing capability of the chimericantibodies in a human breast cancer model, T47D cells were treated with1 ug/ml mAb for 30 min prior to PRL stimulation. At the same time,additional cell samples were incubated with antibody alone to examineany potential agonism gained through chimerization of the antibodycandidates. As can be seen in FIG. 12, all chimeric antibodies retainedtheir ability to block PRL-induced signaling in T47D while onlychXHA.06.983 showed detectable induction of PRLR signaling (a small butreproducible effect) represented by phospho-PRLR and phospho-Stat5.

Determination of Antibody Effect on ERK1/2 Phosphorylation

Selected Human Engineered™ antibodies were tested for their ability toinhibit PRLR-induced ERK1/2 phosphorylation, as described below and inExample 5 above.

Following a 5 hour serum starvation, T47D cells were seeded inmicrotiter plates in complete growth medium for 24 hours at 37° C. Cellswere washed twice with phosphate buffered saline (PBS) and incubatedwith antibodies diluted in serum-free media containing 0.1% BSA for 30minutes at 37° C. The final starting concentration of the antibodies was40 ug/ml. Media was removed and prolactin diluted in serum-free mediacontaining 0.1% BSA was added to a final concentration of 30 ng/ml.Cells were incubated with prolactin for 30 minutes at 37° C. followed bytwo washes with ice cold PBS. Standard lysis buffer containingdetergents, chelators, and various protease and phosphatase inhibitorswas added to generate cell lysates. The levels of phosphorylated ERK1/2(pERK1/2) were measured using standard ELISA according to instructionsof DUOSET® IC Phospho-ERK1/ERK2, R&D Systems, Inc. Results of arepresentative assay are displayed in FIG. 13.

Example 15 A. Ability of mAb Candidates to Mediate ADCC AgainstPRLR-Expressing Target Cells

One of the intended mechanisms of action of anti-PRLR mAb is the abilityto mediate antibody dependent cellular cytotoxicity (ADCC). In order toassess the capacity for ADCC of candidate antibodies, the T47D cellswere employed as PRLR-expressing breast epithelial targets. As shown inFIG. 14, two of the chimeric anti-PRLR mAbs are capable of inducing ADCCmediated by purified human NK cells. chXHA.06.275 was demonstrated toinduce approximately 30% specific lysis of target cells within 4 hr. Dueto prolonged generation time of chXHA.06.642, this candidate was notincluded in the original ADCC assays.

B. Anti-PRLR Antibody Effect on Cytokine Levels

Potential cytokine regulation by PRL in breast cancer cells wasinvestigated. In this experiment, MCF7 or T47D cells were exposed to PRLwith or without chXHA.06.642 for a period of 48 hrs. A multiplexsandwich immunoassay from MesoScale Diagnostics was employed in order tomeasure chXHA.06.642 effects on potential PRL-regulated cytokines. Itwas found that PRL induces VEGF secretion from T47D cells, and that thiseffect was completely abrogated with the addition of antibodychXHA.06.642. No significant regulation of IL-1β, IL-6, IL-8, IL-10,IL-12 p70, IFN-γ, or TNF-α by PRL or anti-PRLR mAb was detected in theseexperiments. These results suggest that the PRL/PRLR pathway maycontribute to angiogenesis as well as cell growth and survival in breasttumors and that inhibiting this VEGF-regulatory pathway may be anotherpotential in vivo mechanism of action for an anti-PRLR therapeuticantibody.

C. In Vitro Combination Studies Utilizing Anti-PRLR mAbs andChemotherapeutics

Due to the possibility that a potential anti-PRLR therapeutic antibodymay be administered in conjunction with cytotoxic drug regimens in theclinic, the effects of such combination therapies on cell survival inculture was investigated. To this end, BaF3/PRLR cells were treated withchXHA.06.642 or chXHA.06.275 at various concentrations in parallel withchemotherapeutics for 5 days, after which cell survival was assessedusing CellTiter Glo as a marker of cell number. An array ofclinically-relevant and mechanistically diverse cytotoxic agents wereutilized in this assay: Doxorubicin (an anthracycline Topo IIinhibitor), Taxol (a microtubule stabilizing agent), Fludarabine (ananti-metabolite), and Cisplatin (a platinum-based DNA cross-linkingdrug). It was found that chXHA.06.275 and, to a greater degree,chXHA.06.642, synergizes with Doxorubicin to enhance cell death inBaF/PRLR cells [See FIG. 15]. The resulting differences inchemotherapeutic IC50 values with and without anti-PRLR mAb chXHA.06.642and chXHA.06.275 are summarized in Table 12.

TABLE 12 KLH chXHA.06.642 chXHA.06.275 Cytotoxic Drug (1 ug/ml) (1ug/ml) (1 ug/ml) Doxorubicin 6.44 nM 2.14 nM 2.56 nM Taxol 4.14 nM 2.07nM 2.45 nM Fludarabine 104.6 uM 40.0 uM 93.8 uM Cisplatin 165.5 nM 27.6nM 46.8 nM

D. Anti-PRLR Functional Activity of Anti-PRLR mAb

Antibodies were assessed in target modulation and cell proliferationassays. FIG. 16 depicts the effect of 2 concentrations of chXHA.06.642and Human Engineered™ antibodies he.06.642-1 and he.06.642-2, onPRL-induced Stat5 phosphorylation in T47D cells. Both retained potentantagonistic properties as evidenced by complete p-Stat5 signalabrogation. Additionally, neither antibody displayed any agonisticactivity or cells treated with mAb alone. Similar results were found forchXHA.06.275 variants. Thus, the Human Engineering™ did not impact theoverall antagonistic or agonistic qualities of these antibodies.

The BaF/PRLR cell proliferation assay was utilized in order to determinerelative IC50 values of anti-PRLR chimeric and Human Engineered™antibodies [See FIG. 17]. As a result of these experiments, all HumanEngineered™ antibodies had approximately equivalent potencies to murinecounterparts.

Example 16 Evaluation of Anti-Tumor Activity of Anti-PRLR Antibody in aNb2-C11 Rat Lymphoma Model

A single-dose PD study was conducted with chXHA.06.642 in the Nb2-C11tumor xenograft model to determine whether the anti-PRLR mAb could reachthe tumor and block signaling. Antibody chXHA.06.642 was used in thisstudy based on its affinity to rat PRLR (described above). The PD markermonitored was p-STAT5, a downstream mediator of PRLR signaling which canbe detected using immunoblot or IHC methods. Since baseline p-STAT5levels in Nb2-C11 tumor xenografts were too low to adequately detect,mice received exogenous ovine (o)PRL stimulation to increase baselinep-STAT5 levels, and thus provide a more suitable dynamic range.

Induction of p-STAT5 was detected by Western and IHC analyses inNb2-Cc11 tumor bearing animals injected with ovine PRL as compared withthe control injected with saline [See FIGS. 18A and B]. Inhibition ofp-STAT5 was observed in mice treated with 10 mg/kg chXHA.06.642 48 hoursprior to oPRL injection and not in the KLH IgG1 treated control animals.

To determine whether inhibition of PRLR signaling correlates withNb2-C11 tumor growth inhibition, a multi-dose efficacy study wasperformed with once weekly administration of chXHA.06.642 [See FIGS. 19Aand B]. Dosing was initiated 4 days post cell implantation (beforetumors were palpable) with chXHA.06.642 or KLH IgG1 at 10 mg/kg, or asaline control. Four weekly intraperitoneal doses of mAb were given. Themodel employed a conditional survival or time to progression endpoint,as these tumors invade the muscle and are thus difficult to measureaccurately with calipers. Tumors in the chXHA.06.642 treated group werenot detected until about 11 weeks post implantation (7.5 weeks after the4th mAb dose), when two of the 15 animals in this group succumbed totumor burden. The median survival in the saline and KLH IgG1 controltreated groups was 20 days post cell implantation (p<0.0001), at whichpoint animals were euthanized due to tumor burden. Animals in thechXHA.06.642 treated group gained body weight while the control animalsmaintained or lost body weight, presumably due to disease burden.

A second efficacy arm was included in this study, in which animals withestablished tumors were enrolled in the study 12 days post cellimplantation [See FIGS. 20A and B]. Mean tumor volumes were 135 mm atthe time of dosing initiation. Animals were dosed intraperitoneally with10 mg/kg once weekly with either chXHA.06.642 or KLH IgG1 controlantibodies for 2 doses. Tumors appeared to fully regress by two daysafter the 2^(nd) dose (3 weeks post-implantation). However,approximately two weeks later tumors started to reappear in the mice. Incomparison, the KLH IgG1 control animals had a heavy tumor burden, whichhad a mean volume of >600 mm³. Since these tumors grow directly into themuscle, mean tumor volume may be larger than that recorded by calipermeasurements. The median survival in both the saline and KLH IgG1 groupswas 23 days post-cell implantation (p=0<0.0001). As in the initialstudy, the animals in the chXHA.06.642 treated group gained body weightwhile the control animals maintained or lost body weight. Thus, thechXHA.06.642 mAb was effective against not only a low number of tumorcells (treatment initiation 4 days after implant), but was also able tocompletely regress the aggressive established Nb2-C11 tumors for morethan 2 weeks.

The Nb2-C11 model has demonstrated that an anti-PRLR mAb has the abilityto effectively target antigen-expressing tumors in vivo, inhibitPLR-driven signaling within the tumor, and induce a measurable outcomein tumor burden, even when the animal has an aggressive establishedtumor.

Example 17 Human Breast Carcinoma T47D Model for PD Assessment

A single dose PD study was performed in vivo with antibody chXHA.06.642tested in Example 16 using breast carcinoma T47D cells. The ability ofoPRL to stimulate PRLR signaling as well as the ability of chXHA.06.642to inhibit this signaling in vivo was evaluated. Tumor-implanted animalsreceived an intraperitoneal injection of saline, KLH IgG1 control mAb orchXHA.06.642, and 48 hours later received either saline or 20 ug oPRL bybolus injection. Forty minutes later tumor tissues were collected. Asignificant induction of p-STAT5 was observed in tumors from the oPRLbolus treated animals, but not in the saline control animals, asassessed by Western blotting, although it is slightly more variable byIHC analysis (FIG. 21). Levels of p-AKT and p-ERK were not increased byoPRL stimulation in vivo. Significantly, treatment with chXHA.06.642,but not the KLH IgG1 control, demonstrated strong inhibition of p-STAT5induction after oPRL bolus injection. IHC analysis generally confirmsthis result. Significantly, p-STAT5 was inhibited in tumors in 4 out of4 chXHA.06.642 treated animals by both Western blotting and IHCanalyses.

Example 18 PRLR Expression and Correlation of PRLR Expression with ERand Her2-neu Expression

In normal tissues, PRLR expression, as quantified by RT-PCR, is highestin breast and uterus followed by kidney, liver, prostate, and ovary.Levels of PRLR mRNA are lowest in the trachea, brain, and lung (Pierce SK, et al., J Endocr; 171 (1):R1-R4 (2001)).

Immunohistochemical (IHC) analyses may be carried out as follows. Frozentissue samples from cancer patients are embedded in an optimum cuttingtemperature (OCT) compound and quick-frozen in isopentane with dry ice.Cryosections are cut with a Leica 3050 CM mictrotome at thickness of 5μm and thaw-mounted on vectabound-coated slides. The sections are fixedwith ethanol at −20° C. and allowed to air dry overnight at roomtemperature. The fixed sections are stored at −80° C. until use. Thetissue sections are retrieved and first incubated in blocking buffer(PBS, 5% normal goat serum, 0.1% Tween 20) for 30 minutes at roomtemperature, and then incubated with the cancer-associatedprotein-specific monoclonal antibody and control monoclonal antibodiesdiluted in blocking buffer (1 μg/ml) for 120 minutes. The sections arethen washed three times with the blocking buffer. The bound monoclonalantibodies are detected with a goat anti-mouse IgG+IgM (H+L)F(ab′)2-peroxidase conjugates and the peroxidase substratediaminobenzidine (1 mg/ml, Sigma Catalog No. D 5637) in 0.1 M sodiumacetate buffer pH 5.05 and 0.003% hydrogen peroxide (Sigma cat. No.H1009). The stained slides are counter-stained with hematoxylin andexamined under Nikon microscope.

Monoclonal antibody against a cancer associated protein (antigen) isused to test reactivity with various cell lines from different types oftissues. Cells from different established cell lines are removed fromthe growth surface without using proteases, packed and embedded in OCTcompound. The cells are frozen and sectioned, then stained using astandard IHC protocol. The CellArray™ technology is described in WO01/43869. Normal tissue (human) obtained by surgical resection arefrozen and mounted. Cryosections are cut with a Leica 3050 CM mictrotomeat thickness of 5 μm and thaw-mounted on vectabound-coated slides. Thesections are fixed with ethanol at −20° C. and allowed to air dryovernight at room temperature. PolyMICA™ Detection kit is used todetermine binding of a cancer-associated antigen-specific monoclonalantibody to normal tissue. Primary monoclonal antibody is used at afinal concentration of 1 μg/ml.

In order to investigate the incidence of PRLR expression and itscorrelation with ER and Her2-neu expression, 122 invasive breast cancerpatient samples were evaluated using immunohistochemistry (IHC).Overall, 62/122 (50%) of the samples expressed PRLR, 58/122 (47%)expressed ER, and 32/122 (26%) expressed Her2-neu. Ninety-six (78%) ofthe samples consisted of invasive ductal carcinoma, of which 48 (50%)expressed PRLR. Among these samples, 24/48 (50%) also expressed ER+ and13/48 (26%) also Her2-neu.

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

1. An isolated antibody that binds the extracellular domain of PRLR ofSEQ ID NO: 2 with an equilibrium dissociation constant (K_(D)) of 10⁻⁶ Mor lower and that comprises (a) the Complementarily Determining Regions(CDRs) set forth at positions 24 through 38, positions 54 through 60,and positions 93 through 101 of the amino acid sequence of SEQ ID NO: 88and (b) the CDRs set forth at positions 31 through 35, positions 50through 66, and 99 through 113 of SEQ ID NO:
 90. 2. The antibody ofclaim 1 wherein the antibody is a chimeric antibody, a humanizedantibody, a human engineered antibody, a human antibody, a single chainantibody or an antibody fragment.
 3. The antibody of claim 1 thatcomprises a constant region of a human antibody sequence and one or moreheavy and light chain variable framework regions of a human antibodysequence.
 4. The antibody of claim 3 wherein the human antibody sequenceis an individual human sequence, a human consensus sequence, anindividual human germline sequence, or a human consensus germlinesequence.
 5. The antibody of claim 1 wherein the heavy chain constantregion is a modified or unmodified IgG, IgM, IgA, IgD, IgE, a fragmentthereof, or combinations thereof.
 6. The antibody of claim 5 wherein theheavy chain constant region is a modified or unmodified IgG1, IgG2, IgG3or IgG4.
 7. The antibody of claim 1 that has an equilibrium dissociationconstant (K_(D)) of 10⁻⁷, 10⁻⁸ or 10⁻⁹ M or lower to PRLR.
 8. Theantibody of claim 1 wherein the light chain constant region is amodified or unmodified lambda light chain constant region, a kappa lightchain constant region, a fragment thereof, or combinations thereof. 9.The antibody of claim 1 that inhibits PRLR intracellularphosphorylation.
 10. The antibody of claim 1 that inhibits the inductionof Stat5 phosphorylation.
 11. The antibody of claim 1 that inhibits theproliferation of a breast cancer cell.
 12. The antibody of claim 1 thatis conjugated to another diagnostic or therapeutic agent.
 13. Theantibody of claim 1 that is purified to at least 95% homogeneity byweight.
 14. A pharmaceutical composition comprising the antibody ofclaim 13 and a pharmaceutically acceptable carrier.
 15. A kit comprisinga therapeutically effective amount of an antibody of claim 1, packagedin a container, said kit containing a second therapeutic agent, andfurther comprising a label attached to or packaged with the container,the label describing the contents of the container and providingindications and/or instructions regarding use of the contents of thecontainer to treat breast cancer.
 16. The kit of claim 15 wherein thecontainer is a vial or bottle or prefilled syringe.
 17. An isolatedantibody that binds the extracellular domain of PRLR comprising avariable light chain amino acid sequence SEQ ID NO: 88, and a variableheavy chain amino acid sequence of SEQ ID NO:
 90. 18. The antibodyaccording to claim 1 that binds the extracellular domain of human PRLRwith an equilibrium dissociation constant (K_(D)) of at least 10,000 to15,000 fold lower than the extracellular domain of murine PRLR.
 19. Theantibody according to claim 1 that binds the extracellular domain ofhuman PRLR, the extracellular domain of murine PRLR, and theextracellular domain of rat PRLR.
 20. The antibody according to claim 19that binds the extracellular domain murine and rat PRLR with anequilibrium dissociation constant (K_(D)) of 10⁻⁶ M or lower.
 21. Theantibody of claim 20 that binds the same epitope as the heavy chain andlight chain variable regions of the he.06.642-2 antibody.